Precise management of crop nitrogen nutrition is essential to maximize yields while limiting pollution risks. For several decades, the critical nitrogen (N) dilution curve - relating plant biomass (W) to N concentration (%N) - has become a key tool for diagnosing plant nutritional status. Increasing number of studies are being conducted to parameterize critical N dilution curves of a wide range of crop species in different environments and N-fertilized conditions. A global synthesis of the resulting data is lacking on this topic. Here, we conduct a systematic review of the experimental data collected worldwide to parametrize critical N dilution curves. The dataset consists of 36 papers containing a total of 4454 observations for 19 major crop species distributed in 16 countries. The key variables of this dataset are the W and %N collected at three or more sampling times, containing three or more fertilizer N rate levels. This dataset can guide the development of generic critical N dilution curves, helps scientists to identify factors influencing plant N status, and leads to the formulation of more robust N recommendations for a broad range of environmental conditions.  However, the over-application of N fertilizers to field crops has a significant impact on the environment through release of greenhouse-gas emissions and air pollutants3, groundwater pollution, and eutrophication of freshwater4 and marine ecosystems5. Increasing N use efficiency could reduce the footprint of N fertilization in agricultural systems while ensuring a high level of production. This goal could be achieved through more precise N rate recommendations and a better application schedule adapted to the crop N requirements.Nitrogen (N) is one of the most limiting factors for agricultural productivity, and mineral N fertilizers represent a key input for many cropping systems worldwide6, plant growth and N demand7. Nitrogen fertilization is often either insufficient (under high fertilizer prices) or excessive when growers adopt conservative overfertilization strategies8. Therefore, for improving N management and recommendation guidelines, both soil and plant processes should be considered for defining optimal N fertilization rates and application schedules.Defining the optimal N fertilization is a daunting task, due to the large uncertainties in predicting soil N supply9. This function is very useful to conduct crop N diagnosis because it allows the computation of the N nutrition index (NNI), defined as the ratio of the actual %N (%Nact) to the value of %Nc corresponding to the observed value of W (Wact). A NNI value close to 1 indicated N sufficiency, while a NNI below or above 1 reveal N deficiency and excessive N status, respectively.Improving our understanding of the co-regulation of N uptake by the availability of N from the soil and plant N demand is relevant to define the overall critical N plant concentration (%Nc), herein defined as the minimum plant N concentration (%N) required to achieve maximum crop mass (W) for a given range of crop species in different environments and N-fertilized conditions. This critical value %Nc is known to decline over time as W increases, and the relationship between %Nc and W defines a function named “critical N dilution curve”Triticum aestivum L.)10,winter oilseed rape (Brassica napus L.)11, maize (Zea mays L.)12, potato (Solanum tuberosum L.)13, rice (Oryza sativa L.)14, and tall fescue (Festuca arundinacea Schreb.)15, among other crops. These curves have been parametrized using either data collected in single experiments or data set pooled from multi-year and multi-sites experiment networks. These curves are now used by many agricultural scientists and engineers for improving fertilization guidelines based on NNI. Many investigations were recently conducted to develop site-specific critical N dilution curves in different parts of the world for many crop species15. However, the scientific value of such local critical N dilution curves is debatable because these curves were parametrized using a small number of local data, leading to high uncertainty and to a risk of false discoveries16. Although sensitivity analyses were performed in some of these studies, they generally suffer from inadequate protocols and methods17. Thus, there is a need for generic and robust critical N dilution curves valid across a large number of crop species in different environments and N-fertilized conditions18. To date, any attempt to develop generic critical N dilution curves has been hampered by the lack of a reliable large-scale dataset. Yet, a global dataset including a large number of pairs of W and %N values is required to drive research efforts on N plant status diagnosis.Critical N dilution curves were established for several major field crops, including wheat  using the following keywords: ‘Nitrogen dilution curve’ & ‘Nitrogen nutrition index’ & ‘Critical nitrogen concentration’ & the name of each crop. The search was performed in the scientific databases ‘ScienceDirect’, ‘Scopus’, and ‘Science Citation Index (Web of Science)’. A last update on this revision to conclude the dataset and include any potential new studies was executed during January 2022. The overall selection process was conducted using the R package 20. The main crop species, papers, number of observations and geographical distribution of the data are presented in Fig. Lolium multiflorum Lam.) 174, broomcorn millet (Panicum Miliaceum L.) 144, cotton (Gossypium hirsutum L.) 80, fodder beet (Beta vulgaris L. ssp. vulgaris) 72, hybrid ryegrass 294, maize 1530, oat (Avena sativa L.) 70, perennial ryegrass (Lolium perenne L.) 19, potato 89, rescue grass (Bromus catharticus H.B.K.) 30, rice 709, sorghum (Sorghum bicolor L.) 95, sugarcane (Saccharum officinarum) 112, sunflower (Helianthus annuus L.) 67, sweet potato (Ipomoea batatas) 120, tall fescue 346, timothy grass (Phleum pratense L.) 256, wheat 234 and white cabbage (Brassica oleracea L.) 13 observations in 16 countries , together with relevant details from each study . If the data were not available in table format, information was retrieved from figures. Data extraction was assisted using the R package juicr21, available at 10.6084/m9.figshare.19105049.v1, and which includes the following files:“NNI_Database.csv” includes the data.“Summary of the database.docx”, includes a summary of the dataset (meta-data), defining each column, trait collected in the data and the units for each variable.“List of references.docx”, presents all the references of the publications included in the dataset.“Figures_NNI.zip”, includes all the codes to build the figures of this study.The data are accessible on the figshare repositoryThe “NNI_Database” contains all the information collected on this systematic analysis. The “Summary of the database” presents a description of the “NNI_Database” file with the information separated into three categories:Category I, general details about the dataset, comprising information for author and publication year, and DOI or other identification for each study included in the dataset.−1), crop material (varieties/hybrids).Category II, relevant to the study, defining species, country, experimental design, years of study, fertilizer N rates levels  and plant N concentration (%N), number of observations per identification number (ID). All the information of W and %N are reported in dry matter basis, as expressed in the data collected from those respective studies.Table Data of plant %N and W are presented in Fig. 18 reported negligible differences across studies. This result revealed that it is more relevant to fit generic critical N dilution curves from a large set of studies covering different environments rather than fitting individual curves to local data. Below, we show that our dataset can be used to establish more universal critical N dilution curve than those generally parametrized from local data.Observed values of W and %N are typically used to fit critical N dilution curves. Fitted curves can help to delineate situations of luxury (excess of N), sufficiency, and deficient (lack of N) plant N status. A recent review of critical N curves obtained for maize cropet al.22. The fitted curve for the field crop can be then compared with a reference curve available in the scientific literature. Lastly as the final part of the second step, an independent dataset can be utilized to assess the plausibility of the NNI values derived from the fitted curve compared to the NNI values derived from the reference critical N dilution model established in the literature. Details of all steps are provided below.To demonstrate the practical value of the dataset, data collected can be used to parametrize a generic critical N dilution curve for the given field crop. After the step on checking for outliers, the field crop W and %N data is used to fit a critical N dilution curve using the Bayesian modeling approach proposed by Makowski 23 and with boxplots, as summarized in the Supplementary material. Any observation with W or %N beyond the threshold of 1.5 difference for the IQR to third quartile was pinpointed as a potential outlier for each trait documented for those studies  based on the interquartile range (IQR) rule detection methodLastly, an overall N responsiveness  was calculated to portray the variation on both W and %N obtained from this dataset due of differential N availability Fig. . The N rNc = A1 × WA2−, where A1 and A2 are two parameters22. The modeling and validation of the critical N dilution curve were performed into four phases:(i)12 for maize crop. First, the data were filtered to include plant W above 1 Mg ha−1. In addition, dates of measures were selected to include maize vegetative and reproductive growth until silking plus 25 days .A pre-processing of the data was conducted following the approach used by Plénet and Lemaire(ii)et al.22. A Markov chain Monte Carlo algorithm (MCMC) was implemented using the R package rjags24. The algorithm was first run with three chains of 50,000 iterations each. Convergence was achieved approximately near 50,000 iterations according to visual inspection of the trace plots and Gelman-Rubin diagnosis. These first 50,000 samples were discarded as “burn-in”, and the algorithm was run again during 100,000 additional iterations to determine the posterior medians and 95% credible intervals of A1 and A2 for maize crop.A Bayesian hierarchical model was fitted to the data following the procedure defined by Makowski (iii)−1 to the maximum value of W in the dataset. Median and 95% credible intervals were determined and plotted against the reference curve established for maize crop by Plénet and Lemaire12 12. This reference N dilution curve was derived from five studies located in France. To avoid any bias due to the use of the same data for fitting and testing, an independent dataset of four maize studies27 was used to compute NNI using the two fitted critical N dilution curves . Error metrics describing the agreement between NNI values were computed using the metrica package28. The root mean square error (RMSE) and mean absolute error (MAE) are standard measures of prediction accuracy, quantifying the average magnitude of the errors in the predictions. The concordance correlation coefficient (CCC) is a measure of both accuracy and precision of the model, quantifying the agreement between an estimated and a reference value. From these metrics, the CCC reflected a strong agreement between both models (CCC = 0.96) and both the RMSE (RMSE = 0.053) and MAE (MAE = 0.046) confirmed that the critical N dilution curve developed with this dataset is very similar to the well-established reference curve12 available for maize , we found that the parameters of the model (A1 and A2) did not differ significantly Fig. . For theIn the future, our dataset could be easily updated with data generated by new studies and, also, with previous studies where data were not available. Studies to be incorporated in this global dataset may be sent to the corresponding author . The goal of our global initiative is expand the dataset by including more data and involve more collaborators. The ambition is the make the largest possible amount of W and %N data available and stimulate the development of reliable critical N dilution curves. In addition to %N, this database could be expanded in near future to cover other nutrients such as phosphorus, sulfur, and potassium, and their interaction with other environmental factors such water stress. Such a collaborative approach may set a milestone in plant physiology and nutrition from which more universal and reliable models could be developed to improve fertilizer management practices and reduce their environmental footprint.Supplementary Information


The cyclists had blood drawn 2 h before and 1 h after the race to assess CK and MB, then the change in pre- and post-race difference was calculated (ΔCK and ΔMB). Each cyclist’s performance time was recorded and the time difference from the winner was calculated (TD). The cyclists’ aerobic capacity was assessed during the incremental test, which determines maximal oxygen uptake and maximal aerobic power. It was observed that 1 h after the cycling race, CK  and MB  increased, compared to pre-race values. Post-race CK increased only in road cyclists, while post-race MB increased only in mountain bike cyclists. Smaller TD were found for lower ΔMB in road cyclists but for higher ΔCK in mountain bike cyclists.The aim of this study was to determine if 1 h after a cycling race, changes in plasma creatine kinase activity (CK) and myoglobin concentrations (MB) differ between mountain bike and road cyclists and if these changes show any correlation with race performance. Male mountain bike cyclists ( Mountain bike (MTB) cycling in the Olympic format (XCO) is a highly intensive race lasting approximately 1 h 30 min, and is performed on technically demanding courses. During competition, the average heart rate exceeds 90% maximal heart rate , and powAside from differences in duration and exercise intensity, competitive road and MTB cycling also differ in muscle activity patterns. Existing literature has noted that the difficult terrain conditions of MTB cycling can elicit significant eccentric muscle contractions for shock attenuation and handling, particularly during downhill sections ,3,4, andThe differences in road cycling and MTB racing formats  can also be reflected in pacing strategy, in which road cyclists show more linear and uniform pacing and power output  than MTBCreatine kinase is a dimeric globular protein consisting of two subunits with a molecular mass 43–82 kDa for each subunit. It buffers cellular ATP and ADP concentrations by catalyzing the reversible exchange of high-energy phosphate bonds between phosphocreatine and ADP produced during contraction ,16. InteSome studies show that blood CK activity is highest approximately 24 h after eccentric exercise  and afteUnderstanding the relationship between potential changes in post-race CK activity and MB concentration and real-world race performance can have several practical implications. Previous studies have demonstrated a strong relationship between winning performance in road cycling and MTB and maximal oxygen uptake, power output at lactate threshold, and gross mechanical efficiency ,27. ThesThe aim of this investigation was to, therefore, determine: (1) if 1 h after the cycling race there would be any significant changes in CK activity and MB concentration, (2) if the changes in CK activity and MB concentration differ between competitive road and MTB cyclists, and (3) if these biochemical markers show a relationship with race performance.It was hypothesized that competition in the road and MTB race would result in greater CK activity and MB concentration 1 h after the race, and that the elevated levels of these biochemical markers would be negatively associated with race performance.n = 14) and MTB (n = 11) male cyclists. All participants were included in the same racing age category: under 23 (U23). No differences in baseline anthropometric measurements were observed between the two groups .D) was calculated as the time loss to race winner, and as a measure of race performance.The MTB cyclists competed in cross-country Olympic (XCO) race format with the start and finish located at 320 m above sea level. The MTB race consisted of six laps, each lap had a distance of 4.8 km and 180 m of elevation, and the total distance during the race was 28.8 km and 1080 m of elevation. The road cyclists competed in a one-day mass start race with the start and finish at 210 m above sea level. The road race consisted of eight laps, each lap had a distance of 17.5 km and 160 m of elevation, and the total distance during the race was 140 km and 1280 m of elevation. During races, both road and mountain bike cyclists drank isotonic carbohydrate drinks  and water, and consumed carbohydrate energy gels . Each cyclist drank about 200 mL of drink for each lap. During the entire race, MTB cyclists consumed two gels, while road cyclists consumed four gels (because their race was longer). The drinks were in bottles mounted on bicycles, while gels were in the pockets of cyclists shirts. The cyclists could refill their drinks and gels by driving through the buffet zone, marked on the race route. Both groups of cyclists provided the same type of carbohydrate drinks and energy gels. As demonstrated by Valentine et al. , it is a change by 72.2 [u∙L−1], but it did not reach the level of statistical significance. However, CK in the road cyclists group increased from 267.1 to 336.9 [u∙L−1], a change by 69.8 [u∙L−1], which achieved statistical significance.Study Limitations. The main limitation of the presented study was the requirement of taking biochemical measurements no later than 1 h after the race, because after this time, the cyclists returned to their homes. According to the previously cited literature, in the case of MB measurement, it was sufficient time to obtain the peak values. However, in the case of CK, it was certainly too short a time to measure the peak. The second limitation of the study was the collection of blood samples only once after the race, as it would be beneficial to collect a few blood samples during the first hour. The third limitation is the smaller size of the group of mountain bike cyclists  in the MTB and road race, respectively.


Black men who have sex with men (MSM) experience disproportionate rates of HIV infection in the USA, despite being no more likely to engage in sexual risk behaviors than other MSM racial/ethnic groups. HIV pre-exposure prophylaxis (PrEP) has been shown to reduce risk of HIV acquisition; however, rates of PrEP use among Black MSM remain low. Clinical, psychosocial, and structural factors have been shown to impact PrEP use and adherence among Black MSM. Care coordination of HIV prevention services has the potential to improve PrEP use and adherence for Black MSM, as it has been shown to improve HIV-related care outcomes among people living with HIV.Client-centered care coordination (C4) is a multi-level intervention designed to address clinical, psychosocial, and structural barriers to HIV prevention services for Black MSM within HPTN 073, a PrEP demonstration project among Black MSM in three cities in the USA. The current study examined the implementation process of C4, specifically investigating the activities, cost, time, and outcomes associated with the C4 intervention.On average, participants engaged in five care coordination encounters. The vast majority of care coordination activities were conducted by counselors, averaging 30 min per encounter. The cost of care coordination was relatively low with a mean cost of $8.70 per client encounter.Although client-centered care coordination was initially implemented in well-resourced communities with robust HIV research and service infrastructure, our findings suggest that C4 can be successfully implemented in resource constrained communities. Black men who have sex with men (MSM) remain disproportionately affected by the HIV epidemic. In 2019, Black MSM comprised more than one-third of new HIV infections in the USA , 4.Clinical, psychosocial, and structural factors are associated with PrEP use and adherence among Black MSM. In prior studies, Black MSM with a history of previous STIs, substance use, and high perceived HIV risk are more likely to use PrEP –7, whileCare coordination has been shown to improve adherence to anti-retroviral (ARV) medications among people living with HIV, but few studies have been conducted that have implemented care coordination to support adherence to ARVs for PrEP –13. CareN = 226) in three cities including Chapel Hill, NC, Los Angeles, CA, and Washington, DC. A full description of the methods used in HPTN 073 is described in the primary outcome paper [The C4™ intervention was implemented in HIV Prevention Trials Network (HPTN) 073, a PrEP demonstration project among Black MSM  as well as any care coordination that occurred between in-person scheduled study visits. Care coordination was not mandatory; thus, even if a care coordination-related need was identified either by the participant or the coordinator, care coordination could be declined by study participants at any time. The C4 process for the study is outlined in Fig. Study team members from each research site completed a three-day training on two main components of C4™. The first component of the training was a foundational grounding in self-determination theory (SDT) provided by Geoffrey C. Williams (University of Rochester Department of Medicine) and Christopher Niemic —two experts in SDT and its application to health promotion. SDT is a social-psychological theory of how social environments can be optimized to support human motivation for pro-health behaviors and is the organizing theoretical framework for C4™. Second, the University of Rochester Center for Community Practice—a CDC Part II Prevention Training Center at the time—provided training on Comprehensive Risk Counseling & Services (CRCS), which is a prevention case management program model in the CDC’s compendium of evidence-based interventions. The CRCS training included tailored content for how to apply the SDT concepts in the delivery of CRCS with Black MSM as well as skills-building on the use of CRCS strategies to support issues related to PrEP use and adherence. All participants were provided with a standardized C4™ implementation manual and access to on-demand technical assistance by the second author.Care coordination activities were measured using an adapted version of the Care Coordination Measurement Tool (CCMT) . The CCMgreater than 5 min of coordination of services. The tool was designed to ensure time spent on care coordination activities was documented to the nearest minute see Fig. . The meaPersonnel costs were the costs associated with C4 components of the intervention. Personnel costs were calculated using the current median (mean) hourly rates for each occupation conducting C4 based on their metropolitan area using the US Bureau of Labor Statistics .Statistical analysis consisted of descriptive statistical analysis: means and standard deviations, frequencies of items collected from the CCMT, and cost calculated using the US Department of Labor Statistics. All analysis was performed using the R software, version 3.4.1.N = 76), Chapel Hill (N = 75), and Washington DC (N = 75). Of those participants, 86.3% identified as African American, and 7.5%, 3.5%, and 0.4% identified as Afro-Latino, Afro-Caribbean, and African, respectively. During the study, 78.8% initiated PrEP; of those who initiated PrEP, 70.8% initiated within the first 30 days of study enrollment. One-third of participants (29.5%) were below the US poverty line with an annual income less than $10,000 [An approximately equal number of participants were recruited across the three cities: Los Angeles , followed by Los Angeles 33.2% (n = 377) and Chapel Hill 18.7% (n = 212) sites. The care coordination encounters covered a range of health and social support topics. Table n = 492, 43.3%), sexual health services , or social services  such as housing, food, and clothing.During the study, the total number of care coordination encounters was 1,135, with an average of 5.0 encounters per participant. The highest proportion (48.1%) of encounters occurred in Washington DC (Care coordination activities were primarily conducted by counselors (96.3%), followed by nurse practitioners (1.8%) and social workers (1.0%). Other healthcare workers who performed care coordination were registered nurses, physician assistants, family therapists, and HIV risk reduction counselors; however, each of these roles accounted for < 1% of care coordination encounters. The time that health care workers spent on care coordination activities to address the needs of their clients varied from 5 min per encounter to > 50 min, with most encounters taking between 30 and 39 min.n = 383; 28.8%), chart review , and telephone discussions directly with the participant being served . The activities performed to fulfill care coordination needs varied across sites. In Los Angeles, developing or modifying an existing care plan  was the most common activity performed. In Washington DC, developing and modifying an existing written prevention plan  was also most performed. Chapel Hill differed from both sites, with telephone communication directly with participants  and client-focused research  were recorded as the most common activities performed to fulfill the participant’s care coordination needs. A full description of the activities performed at each site is listed in Table A total of 1,331 recorded activities occurred over the 1,135 encounters. The most frequently performed activities were as follows: developing or modifying an existing written prevention plan , which was followed by referral to a community agency. Other referrals included referral to a primary care office, lab, or specialist , resulting in 531.8 personnel hours. A full breakdown of care coordination activities by personnel type and implementation city can be found in Table A total of 549.6 personnel hours of C4 intervention delivery were logged related to 1,135 C4 encounters. The total personnel costs of care coordination were $9,826, resulting in a mean cost of $8.70 per client encounter. The average time for each encounter was 29 min, which varied by personnel type (range: 17–40 min). The lowest cost per encounter was with an HIV tester ($7.10/encounter) and the highest was with a registered nurse ($21.30/encounter). Most encounters occurred with counselors  or made active referrals to other community agencies for services not offered by the organization . The case workers followed up with clients to assess their access of the services and provide additional support for the services accessed, if needed. As self-determination was an essential philosophy of the intervention, the social determinants of health addressed by in intervention staff we driven by the needs identified by participants. Sexual health and social services, including PrEP adherence counseling, were the most frequent focus of care coordination encounters. Participants also accessed care coordination for mental health, medical services, and substance use treatment. The range of services used by participants illustrates the breadth of service needs for Black MSM. These findings highlight the need for the availability and accessibility of services that can support Black MSM’s psychosocial needs and address the negative impacts of clinical, psychosocial, and structural factors on their engagement in HIV prevention services. These findings supports existing research which argues that to reduce HIV incidence in Black MSM, HIV prevention interventions must address social determinants of health , 19. FurThe broad service encounters resulted in a large portion of time being dedicated to development and modifying care plans and the coordinator researching services to meet the immediate needs of the HPTN 073 participants. These activities suggest that implementation of C4™ requires ongoing knowledge of community supports and services, maintaining relationships with community-based organizations and social service providers, and resources available to support clients in accessing support and auxiliary services . In HPTN 073, the ability to facilitate services was dependent upon availability of services in the community; thus, implementation of C4™ is highly impacted by range of services in the community. Higher resourced communities may be able to facilitate greater access to service needs identified by clients. These findings are similar to research which suggest the availability of community services influences health outcomes, with greater resourced communities having better health outcomes –26.There are numerous costs involved with leveraging a C4 program that are embedded within the research effort and would need to be considered when launching a program in a real-world setting. Particularly in network studies with existing research infrastructure already established, the costs of implementation may seem deceptively small because key program functions are already paid for and not counted within the specific study budget. Specific potential implementation costs are outlined in Table M = $56.08), followed by physician assistants (M = $47.21) and registered nurses (M = $38.27). The average mean hourly wage across personnel type was highest in Los Angeles ($35.65), and similar within Washington DC ($32.49) and Chapel Hill ($31.50). These costs were based on mean personnel costs based on the US Bureau of Labor Statistics and actual costs were not assessed in the study. The implementation of C4™ may be highly variable based on the availability of community resources and labor costs of the available healthcare facility staff. Our study findings suggest that care coordination outcomes were more advantageous in Washington DC site than in Chapel Hill and Los Angeles. Unfortunately, there was no qualitative follow-up with clients to determine why care coordination was more advantageous in certain cities. Further research is needed to determine the impact of community-level factors on the engagement of C4 and how availability of community resources might impact the overall outcome of engagement in C4. It might be due to participant resources outside of the intervention impacted their engagement in C4. Research demonstrates that those who have more resources are more likely to benefit from HIV interventions [Implementation of C4 in this PrEP demonstration study proved to address the holistic needs of Black MSM; however, this intervention is not without limitations. Because C4 was embedded in a network study, the total costs of implementation  costs are not accounted for in this study. However, this study was able to parse out specifically the C4 delivery costs from the overall program costs. While that does not give much in terms of being able to figure out overall program costs, it does have the unique benefit of being generalizable since it looks at minutes of delivery. If a local organization wanted to implement C4, they could look at the specific time commitment/resources needed to add C4 specifically onto their existing program offerings. While personnel costs were relatively low, the expenditures at each site varied based on the differences in salaries and the availability of resources. Furthermore, the study was not designed to evaluate the cost-effectiveness of the intervention; therefore, implementation of this intervention would need to account for indirect costs associated with implementation of C4. Future studies of C4 should determine full intervention costs to determine cost-effectiveness. Personnel mean hourly wage used in C4™ varied by personnel type and location, but it was consistently highest for nurse practitioners ( M = $47. and regiventions .A client-centered care coordination intervention—C4™—was successfully implemented across three cities in the HPTN 073 study. Although client-centered care coordination has been implemented in well-resourced communities with robust HIV research and service infrastructure, our findings suggest that C4™ can also be successfully implemented in lower resourced communities. C4™ program activities can be performed by most staff at organizations, reducing the provider associated costs and burden. Understanding the activities, personnel, cost, and time needed for successful implementation of care coordination interventions is critical administrative evidence for informing future programmatic dissemination, implementation, and stainability.


Frequent high temperature weather affects the growth and development of rice, resulting in the decline of seed–setting rate, deterioration of rice quality and reduction of yield. Although some high temperature tolerance genes have been cloned, there is still little success in solving the effects of high temperature stress in rice (Oryza sativa L.). Based on the transcriptional data of seven time points, the weighted correlation network analysis (WGCNA) method was used to construct a co–expression network of differentially expressed genes (DEGs) between the rice genotypes IR64 (tolerant to heat stress) and Koshihikari (susceptible to heat stress). There were four modules in both genotypes that were highly correlated with the time points after heat stress in the seedling. We further identified candidate hub genes through clustering and analysis of protein interaction network with known–core genes. The results showed that the ribosome and protein processing in the endoplasmic reticulum were the common pathways in response to heat stress between the two genotypes. The changes of starch and sucrose metabolism and the biosynthesis of secondary metabolites pathways are possible reasons for the sensitivity to heat stress for Koshihikari. Our findings provide an important reference for the understanding of high temperature response mechanisms and the cultivation of high temperature resistant materials. Global warming has become one of the serious challenges that mankind cannot ignore. High temperature hazards caused by warming have severely affected crop production and food security. It is estimated that for every 1 °C increase in the average daily temperature, yield losses in rice, maize and wheat will increase by 10–25% [2+ concentration mediated by membrane localized Ca2+ channels [Heat stress (HS) is defined as irreversible damage to plants due to the temperature beyond a physiology threshold level . Rice ischannels ,7. To dachannels .Sogatella furcifera and Nilaparvata lugens responsive genes and specific regulation pathways have been found in rice by omic data [With the development of data analysis methods and the further cost reduction of high throughput sequencing, RNA sequencing (RNA–seq) has become a routine technique in response to the various biotic and abiotic stresses of rice. The amount of HS, cold, cadmium, mic data ,10,11. Tmic data . The clamic data . When comic data ,15. Genemic data . Each nomic data ,18.Weighted correlation network analysis (WGCNA) is a system biology method used to describe gene association patterns between different samples. It can identify gene sets (modules) with similar expression patterns, analyze the relationship between modules and sample phenotypes, draw the regulatory networks between genes in the modules and identify key regulatory genes ,20. The indica variety IR64 and the japonica variety Koshihikari as study materials. IR64 is the parent of modern indica rice varieties. Koshihikari is an elite Japanese variety. Both IR64 and Koshihikari are the most popular indica and japonica rice varieties in the world and are widely grown in various rice producing areas. The transcriptome information of the two genotypes at seven time points after HS was obtained using RNA–seq. Based on these data, we performed WGCNA analysis to explore the highly correlated modules and co–expressed genes. By analyzing the interaction network between the co–expressed genes of the two genotypes and the studies of HS–related genes, the ultimate goal was to identify the hub genes and preliminarily explain the reasons for the different responses of two genotypes to HS.Here, we used the The moderately resistant variety “IR64” and highly susceptible variety “Koshihikari” were provided by the College of Agronomy at Hunan Agricultural University. Rice seedling plants were grown hydroponically in a growth phytotron at 28 °C with a 12 h day/12 h night cycle, and a humidity of 70% to 10 day old stage, then treated with 45 °C for HS.Leaf samples were harvested at seven time points  after HS, and 42 sets of RNA–seq data  were obtained in this study. Both genotypes were based on 0 h (control), and a large number of DEGs were obtained at each time point . The rawThe construction of the WGCNA co–expression network was achieved using the WGCNA package in R version 4.1.1. The average expression of each gene at different time points was calculated, and the genes with no change in expression were filtered out. The expression level of each gene was normalized 0–1, and Pearson’s coefficient was used to calculate the correlation between genes to determine the co–expression similarities of two genes. An appropriate adjacency matrix weight parameter β value was selected to satisfy the precondition of scale free network distribution, and the Topological Overlap Matrix (TOM) was constructed for clustering and segmenting the modules. The relationship between each network module and the sample phenotype were analyzed, so as to select the modules related to the time points.p < 0.05 indicating a statistically significant difference and a correlation of module–trait value cor ≥ 0.5 which indicating a vital module. To directly describe the gene expression in each vital module, we used singular value decomposition in transforming the gene expression data from the gene space to eigengene space, where eigengenes are the unique orthonormal superpositions of the genes for each module.The significance of the different time point modules was compared, with The module eigengene (MM) value can be obtained by analyzing the correlation between the expression of the gene and the corresponding module eigengene. The MM value is essentially a correlation coefficient. If the absolute value of MM is close to 1, it indicates that the gene is highly correlated with the module. The correlation analysis is performed between the expression of the gene and the corresponding phenotype value. The final value of the correlation coefficient is gene significance (GS). GS reflects the correlation between the gene expression and the phenotype data. The higher the GS, the more relevant the gene in the research phenotype. In this paper, we took MM ≥ 0.75 and GS ≥ 0.2 as the criteria to screen the key genes in the vital modules.p < 0.05 was the significance threshold. The cluster and visualization of the key genes were constructed through the MCODE, Centiscape and Protein–Protein Interaction Networks (PPI) plugins of Cytoscape (3.9.0).The key genes found in two genotypes were subject to the Gene Ontology (GO) database and the Kyoto Encyclopedia of Genes and Genomes (KEGG) for function and pathway enrichment analysis; https://shigen.nig.ac.jp/rice/oryzabase/gene/list, accessed on 1 March 2022) and RiceData . Combined with a clustering algorithm, high connectivity genes were defined as known–core genes. The known–core genes were compared with the key genes, and the candidate hub genes of the two genotypes were obtained by K-means clustering.A more comprehensive search for genes with high connectivity in both genotypes was carried out. The terms “heat stress” and “high temperature” were used to screen the related genes studied through the Oryzabase , bisque4 (710 DEGs), darkred (1602 DEGs), cyan (1233 DEGs), black (1195 DEGs), lightsteelblue1 (231 DEGs), paleturquoise (444 DEGs) and grey (83 DEGs) were obtained in IR64 A. Nine mcor ≥ 0.5 and p < 0.05 as the criterion, we found several specific modules with significant correlations for IR64 and Koshihikari. For IR64, the darkred module  at 1 h, the bisque4 module  at 24 h, the black module  at 24 h and the green module  at the late time point showed high correlation with HS  at 2 h, the black module  at 24 h, the plum2 module  at 24 h and the white module  showed high correlations with HS (The preliminary study (unpublished data) shows that DEGs at 4 h post HS between the two cultivars were the lowest at all time points, suggesting that 4 h after HS might be an important critical time point. Thus, we added early (before 4 h) and late (after 4 h) time points to process A,B. Thro with HS . For Kos with HS .The correlation between each gene and eigengene was calculated, the module membership (MM) < 0.75, the gene significance (GS) < 0.2 of genes in the module were deleted, and we finally obtained key genes in the specific module . To furtFor Koshihikari, the grey60 module displayed significant enrichment in ribosome and oxidative phosphorylation. In terms of GO, ATP metabolic process, proton–transporting ATPase activity, rotational mechanism, translation elongation factor activity and cytochrome–c oxidase activity were found. For the plum2 module, protein processing in the endoplasmic reticulum and plant–pathogen interaction on KEGG terms, and small GTPase–mediated signal transduction and GTP binding on GO terms were found. For the black module, the highly enriched genes, mostly on DNA or RNA processes and repair pathways such as spliceosome, RNA transport, RNA degradation, nucleotide excision repair and mRNA surveillance pathway were found. At the white module, the biosynthesis of secondary metabolites, carbon metabolism, citrate cycle (TCA cycle) and carbon fixation in photosynthetic organisms were significantly enriched –D. BasedOsWRKY1, OsWRKY53, OsMYB30, OsWRKY24, OsWRKY28, OsWRKY10, OsMYB55), six heat–shock transcription factor (HSF) genes , a bHLH protein gene (EAT1) and a NAC protein gene (LOC_Os11g03370). In addition, 28 ribosomal protein–related genes  and 10 heat–shock protein (HSPs) genes  were regulatory nodes for HS.Through the Oryzabase and RiceData databases, we obtained 490 genes related to the HS of rice . AccordiCHR745, hsp82B, OsBip1, OsBip2, OsBiP3, OsBiP4, OsDjA7, OsGrp94, OsGSK1, OsHSP1, OsHSP58.7, OsSTN8, OsTT1, OsUBC32, OsUBP6 and PP2A–A), with 19 putative ribosomal proteins and five putative Dnak family proteins included. For Koshihikari, 18 genes were cloned , along with 20 putative ribosomal proteins and two putative Dnak family proteins. Among the candidate–hub genes of IR64 and Koshihikari, 28 genes were identical, which implies that there was a consistent HS response among the genotypes. Overall, two genotypes of rice were found to share some of the same hub nodes for the response to HS; however, different specific regulation responses were also involved.In order to more effectively search for the hub genes’ response to HS, we performed an interaction network analysis of IR64 and Koshihikari key genes with known-core genes, respectively. According to the K-means clustering algorithm, the interaction network was divided into three main modes, and the top 20 genes of each mode were taken as candidate hub genes . As showTo identify features of the 60 candidate hub genes for IR64 or Koshihikari in response to HS, function and pathway enrichments were performed. The GO enrichment results show that the candidate hub genes of IR64 and Koshihikari are enriched to consistent terms. Within BP, “cellular process” and “metabolic process” were the prominent enrichment terms. Within CC, “cell”, “cell part”, “organelle” and “organelle part” were the prominent enrichment terms. Within MF, “binding”, “structural molecule activity” and “catalytic activity” were the prominent enrichment terms A,B. In tGlobal warming is a serious problem. The high frequency and prolonged periods of HS have become a direct threat to the productivity of agricultural crops, including rice ,25. Unde2O2 and irreversible damage to the photosynthetic apparatus [Through the correlation analysis of modules using time points and eigengenes, four modules were selected each for IR64 and Koshihikari. We took the highly correlated genes in the modules as core genes that were used for further biological function and metabolism analysis. In the four modules of the two genotypes, we found that carbohydrate metabolism, energy metabolism and amino acid metabolism were the elements of the metabolic pathways most involved in HS. Carbohydrate metabolism includes starch and sucrose metabolism, amino sugar and nucleotide sugar metabolism, and glyoxylate and dicarboxylate metabolism, among others . Carbohypparatus ,35,36. Mpparatus . Long tepparatus . Overallcis–elements of a given gene in a specific biological environment, affecting the expression of a vast number of downstream genes [cis–elements MYBRS, MYCRS and ABRE (core sequence ACGTGGC) in the promoter region of downstream stress response genes RD22, Gly, RD29B and RD20A [NTL4, an NAC transcription factor gene, forms a positive feedback loop with ROS, causing a sharp increase in ROS at high temperatures, recycling nutrients and metabolites from damaged tissues to the meristem or newly formed leaves, causing local programmed cell death (PCD), thereby enhancing plant survival. The constitutive overexpression of AtWRKY25 and AtWRKY26 could enhance the resistance to HS, as the reaction pathways are intertwined with the interactions of many plant hormones, calcium and ROS [2O2 treatments, salt and infection with a bacterial pathogen, xanthomonas oryzae, which causes leaf blight [HSPs are activated to play protective roles when plants are confronted with various adversities [cis–acting regulatory elements in the promoter region of the HSP genes; the activity of HSFs is also regulated via a feedback loop formed by the physical interaction with HSPs [A large number of HS–related QTLs have been identified and cloned, but efficient aggregation and clustering is lacking. In this study, we obtained 490 genes related to HS, and divided them into 14 clusters with 145 known–core genes. In total, 14 transcription factors (TFs), 28 ribosomal proteins (RPs), and 16 heat shock protein (HSPs) and heat–shock transcription factor (HSF) genes had a strong degree of association in the network of interaction. TFs play a regulatory function through the forming of transcriptional complexes, which bind to local and distal am genes ,40. Whennd RD20A . NTL4, a and ROS . RPs aref blight . Variousf blight ,45. As mersities . An incrersities . The expith HSPs . It has ith HSPs ,49,50. UOsDjA7 and OsGrp94 were common genes in both genotypes. OsDjA7, the first characterized DnaJ gene, is involved in DNA replication and repair through the interaction with the proliferating cell nuclear antigen (PCNA) gene, and also functions in the chloroplast development of rice [OsGrp94 belongs to the HSP90 family protein. HSP90/Gas2/HSP40 could form a caspase–3–related protein complex in rice suspension cells’ response to HS. HSP90/Rac1/RAR1/HSP70 could form one or more protein complexes in rice cells and play important roles in the innate immunity of rice [OsDjA7 and OsGrp94 not only respond to stress, but also function in slowing down or repairing the physiological damage caused by stress. OsGSK1 and OsTT1 were the unique response genes in IR64. OsGSK1 has physiological roles in brassinosteroid (BR) signal transduction pathways and functions in several stress responses, including cold, heat, salt and drought [OsTT1 was obviously selected by environmental temperature, and then functional variation occurred to make the corresponding varieties adapt to their growth environmental temperature. The study of Thermo-Tolerance1 (OgTT1), cloned from African rice (Oryza glaberrima), shows that OgTT1 protects cells from HS through the more efficient elimination of cytotoxic denatured proteins and more effective maintenance of heat–response processes than achieved with OsTT1 [OsALDH5F1 and OsALDH7, and the betaine aldehyde dehydrogenase gene, OsBADH1, were the unique response genes in Koshihikari. ALDH is considered the antidote to reactive oxygen species in organisms. It oxidizes toxic aldehydes into corresponding non–toxic carboxylic acids, maintains the balance of aldehydes and plays an important role in stress physiology [OsBADH1, encoding a key enzyme for the glycine betaine biosynthesis pathway, has a physiological function in the oxidation of acetaldehyde produced by catalase, which is involved in multifunctional mechanisms in response to environmental stresses, including salt, plasmolysis, temperature and light stress [Core genes are the markers for the processes involved in a specific biological response, and hub genes usually play an essential role in gene regulation networks . Based o of rice ,53,54,55 of rice ,57. OsDj drought ,59. Ther drought . During th OsTT1 . Two aceysiology . OsBADH1t stress ,63,64.The GO and KEGG enrichment analyses of the 60 candidate–hub genes unveiled an interesting result in the two genotypes. IR64 and Koshihikari had consistent GO terms and partly common metabolic pathways. However, starch and sucrose metabolism and the biosynthesis of secondary metabolite pathways were the unique metabolic pathways. Starch and sucrose metabolism is the downstream branch of carbohydrate metabolism. When photosynthesis is damaged, starch and sucrose do not only provide carbon energy, but also behave as osmo protectants and compatible solutes to alleviate the negative effects of stress . SecondaIn summary, a gene co–expression network based on WGCNA was constructed using the transcriptome scale changes of the two genotypes under HS. The highlight of this study is that the core genes were systematically screened based on the studied HS research. Through the co–expression modules identified at different time points and the interaction network analysis with known HS related genes, we took the top 20 genes in terms of their degree of connection as candidate hub genes. The analysis of these candidate hub genes found that, compared with IR64, starch and sucrose metabolism and the biosynthesis of secondary metabolites were more significantly enriched in Koshihikari, indicating that starch and sucrose metabolism and the biosynthesis of secondary metabolites might play a critical role in response to HS for Koshihikari. Even though the results have some limitations and further experimental studies are required to verify the function in response to HS, a reliable basis remains for further research into the molecular mechanisms and the mining of key genes in response to HS.


Recent studies in experimental animals found that oral exposure to micro- and nano-plastics (MNPs) during pregnancy had multiple adverse effects on outcomes and progeny, although no study has yet identified the translocation of ingested MNPs to the placenta or fetal tissues, which might account for those effects. We therefore assessed the placental and fetal translocation of ingested nanoscale polystyrene MNPs in pregnant rats. Sprague Dawley rats (N = 5) were gavaged on gestational day 19 with 10 mL/kg of 250 µg/mL 25 nm carboxylated polystyrene spheres (PS25C) and sacrificed after 24 h. Hyperspectral imaging of harvested placental and fetal tissues identified abundant PS25C within the placenta and in all fetal tissues examined, including liver, kidney, heart, lung and brain, where they appeared in 10–25 µm clusters. These findings demonstrate that ingested nanoscale polystyrene MNPs can breach the intestinal barrier and subsequently the maternal–fetal barrier of the placenta to access the fetal circulation and all fetal tissues. Further studies are needed to assess the mechanisms of MNP translocation across the intestinal and placental barriers, the effects of MNP polymer, size and other physicochemical properties on translocation, as well as the potential adverse effects of MNP translocation on the developing fetus. After three quarters of a century of increasing production of and reliance on plastic materials, six billion metric tons of plastic waste have been deposited in the environment —enough tResults from previous in vitro and in vivo studies suggest that ingested MNPs can cross the intestinal barrier to access the circulatory system, and thence the peripheral tissues. Walczak et al. reported the size- and surface-chemistry-dependent uptake of MNPs in multiple in vitro intestinal cell models . LikewisWe therefore hypothesized that ingested nanoscale PS MNPs could translocate across the intestinal epithelium to reach the circulation, and subsequently cross the maternal–fetal barrier of the placenta to reach the fetal circulation and tissues. In the present study, we tested this hypothesis in a pregnant rat model, as detailed below.To assess the placental and fetal translocation of MNPs, we gavaged pregnant  female Sprague Dawley rats with 10 g/kg of a 250 µg/mL suspension of 25 nm carboxyl-modified PS spheres (PS25C) and harvested placentas and fetal tissues after 24 h (GD20) for analysis using enhanced darkfield hyperspectral microscopy (EDHM), which allows for the identification and localization of materials within tissues based on their specific hyperspectral signal. An overview of the study design is shown in Primary carboxyl-modified red fluorescent PS nano-microspheres with sizes of 25 nm (PS25C) were obtained from Thermo, Inc. .Carboxyl-modified red fluorescent FluoSpheres™ were obtained from Thermo, Inc. . The nominal size of the spheres was 20 nm (25 nm according to the manufacturer’s characterization for the product lot used). MNPs from Thermo were diluted in sterile deionized water to the desired starting MNP concentration and vortexed for 20 s.dH) and polydispersity index (PdI), and by electrophoretic light scattering (ELS) to determine zeta potential (ζ) and conductivity (σ) using a Zetasizer Nano-ZS  as previously described [Water suspensions of PS25C used for gavage were analyzed by dynamic light scattering (DLS) for the determination of hydrodynamic diameter  17–18 and randomly divided into control and exposure groups. Exposure group animals were lightly anesthetized with isoflurane gas (5% induction) and gavaged with a bolus dose of MNPs  using a 20-gauge 4-inch stainless-steel ball-tipped animal feeding needle on GD 19. Control animals remained untreated. Rats were monitored after gavage until they had regained consciousness and normal physiological activity . Exposed and control animals were sacrificed 24 h following exposure per approved Rutgers’ IACUC protocols. Maternal tissues, periparturient fetuses and fetal tissues  were harvested, fixed in 10% formalin and embedded in paraffin. Maternal weight, litter number, fetal weight, placental weight, placental efficiency and resorption number were recorded.To prepare specimens for EDHM analysis, thin sections (5 µm) of formalin-fixed, paraffin-embedded placental and fetal tissues were deparaffinized by washing three times for 5 min in xylene, then rehydrated through a series of washes in 100, 95, 70 and 50% ethanol (2–10 min washes each), followed by 2–5 min washes in deionized water and finally washed three times in PBS. Slides were then air dried and mounted with SlowFade Diamond mountant  and a #1.5 coverslip.A CytoViva Enhanced Darkfield Hyperspectral Microscope  was used to identify and visualize PS25C within placental and fetal tissue sections. Enhanced darkfield hyperspectral microscopy (EDHM) allows imaging of nanoscale sample elements based on their scattering or emission properties. The system utilizes a tungsten halogen light  illumination source with a variable power (0–150 W) control, which was set to the full 150 W output for these experiments. Light from this light source is directed to the microscope via a liquid light guide . The light guide is connected to the enhanced darkfield illuminator system, which is mounted onto the condenser mount of the research-grade optical microscope. The illuminator system consists of an annular cardioid oil condenser, which produces highly collimated light at oblique angles with a numerical aperture of 1.2–1.4 and collimating lenses, which together modify the geometry of the source light to closely match that of the system’s cardioid oil condenser. The collimating lenses focus the light onto the condenser to refine and fix the Koehler illumination of the light onto the condenser, allowing the highly oblique darkfield illumination to be focused onto the precise focal plane of the sample without losing the integrity of the Koehler illumination. This indirect oblique illumination allows collection of the reflected or elastically scattered light from the sample, which permits the visual differentiation of objects with refractive indices similar to that of the background.In the EDHM system, the scattered light image from the objective is projected onto a visible and near-infrared (VNIR) diffraction grating spectrograph , which separates the light into distinct wavelengths from 400 to 1000 nm with a spectral resolution of 2 nm. This spectrally resolved light is then projected through a 30 µm slit onto the pixels of a charge-coupled device (CCD) video camera . This camera then sends the data to a highly custom version of ENVI 4.8 hyperspectral image capture analysis software. The hyperspectral image is captured in a pixel row by pixel row line scan using an automated translational stage  with a 10 nm step resolution. The hyperspectral image is then built, pixel row by pixel row.Analysis of resulting hyperspectral images was conducted using a customized version of ENVI 4.8 hyperspectral image analysis software . Each pixel of the hyperspectral image contains the optical spectral response from 400 to 1000 nm of that pixel’s spatial area at a 2 nm spectral resolution. Image analysis was conducted to spectrally map labeled polystyrene particles in the tissue. Scattered light was collected using a 60x oil iris objective . Thousands of individual pixel spectra were captured to create a spectral library from tissues from pups of dams exposed to PS25C nanoparticles. These spectral libraries were then compared to all pixels in the negative control tissue (from pups of control dams) image in a process known as “Filter Spectral Library”. A spectral mapping algorithm known as “Spectral Angle Mapper” (SAM) was then utilized to match pixels to the reference spectrum to spectrally map the polystyrene in tissue.The water suspension of PS25C was analyzed by dynamic light scattering (DLS) and electrophoretic light scattering (ELS), which revealed a monomodal particle dispersion with an average hydrodynamic diameter of 33.02 ± 0.01 nm, polydispersity index of 0.151 ± 0.013, zeta (ζ)-potential of −66.8 ± 1.76 mV and conductivity (σ) of 0.0414 ± 0.0004 mS/cm. Intensity- and volume-weighted size distributions are shown in Hyperspectral images of 4 µm sections of placentas from PS25C-exposed dams are shown in To assess the potential fetal tissue translocation of ingested PS25C, EDHM was used to identify PS25C MNP signals within sections of fetal organs harvested 24 h after oral gavage. Large clusters of PS25C were identified in all gestationally exposed fetal tissues examined, including liver, kidney, lung, heart and brain . The fetThe placenta is a well-known barrier, but also functions as a vascular and endocrine organ to enable fetal development. Because such functions could potentially be impaired by the presence of MNPs, we assessed changes in critical maternal and litter characteristics, including maternal weight, litter size, fetal weight, placental weight, placental efficiency and total number of sites of resorption. The results of these measurements in control and treated rats are reported in Histopathological examination of fetal liver, kidney, lung, brain and heart tissues from three pups from each control and PS25C-treated dam revealed no notable findings in any of the tissues and no notable differences between tissues from pups of treated and untreated dams.The results of this in vivo study confirm our hypothesis that ingested nanoscale MNPs can (1) translocate across the intestinal epithelium to reach the circulation and (2) cross the maternal–fetal barrier of the placenta to reach fetal circulation and tissues. These findings strongly suggest the potential for the maternal–fetal transfer of nanoscale PS MNPs.In this experimental model, the PS MNPs must cross two physiological barriers to reach the fetal compartment: the gastrointestinal tract and the placenta. As described in the Introduction, studies by our lab and others have found that PS MNPs can cross the intestinal epithelium and distribute to multiple organs. Further studies are needed to determine the specific mechanisms involved in MNP translocation across the intestinal epithelium, which could include passive transcellular and/or paracellular diffusion as well as one or more active (energy-dependent) endocytosis mechanisms, recently reviewed by Rennick et al. . Such stThe second physiological barrier required for translocation to fetal tissues is the maternal–fetal barrier, which occurs in the placental labyrinth zone. In rats this consists of four distinct layers: a layer of cytotrophoblasts, a double layer of syncytiotrophoblasts and the fetal capillary endothelium . Because the cytotrophoblast layer is discontinuous and the fetal capillary endothelium is fenestrated, they do not contribute substantially to barrier function. The two continuous, uninterrupted syncytiotrophoblast layers, which are connected at gap junctions through Connexin 26, act functionally as a single syncytial layer to provide barrier function . The appThe presence 24 h post gavage of PS25C within all fetal tissues examined from pups of exposed dams, including liver, kidney, lung, heart and brain , is consA significant decrease in placental weight was also observed in PS25C-treated dams compared to untreated controls, while no significant difference was seen in fetal weights between the groups, resulting in an increase in calculated placental efficiency in the treated dams . HoweverFinally, it is worth noting that simplistic models of MNPs such as the PS used in this study are not environmentally relevant. More environmentally relevant MNPs which are by-products of the thermal, mechanical and photo-induced degradation of polymers in the environment are needed for future toxicological assessment studies. It is well known that the physicochemical properties of nanoscale materials define their cellular uptake and biodistribution ,36. SuchThis is the first study confirming maternal translocation of ingested MNPs to fetal tissues in a mammalian species. More studies are needed, using environmentally relevant MNPs across a range of highly produced polymers, as discussed above, in order to understand the full scope of potential hazards that ingested MNPs might represent. Further studies are also needed to assess the impact of the presence of MNPs in placental and fetal tissues and cells on fetal health and pregnancy outcomes, in order to uncover the mechanisms involved in MNP uptake by the intestine and in MNP translocation across the maternal–fetal barrier of the placenta, and to determine the role of MNP properties such as polymer type, surface chemistry , size and shape on uptake, fetal translocation and health implications.


Seasonal rhythms drive metabolic adaptations that influence body weight and adiposity. Adipose tissue is a key regulator of energy homeostasis in the organism, and its healthiness is needed to prevent the major consequences of overweight and obesity. In this context, supplementation with proanthocyanidins has been postulated as a potential strategy to prevent the alterations caused by obesity. Moreover, the effects of these (poly)phenols on metabolism are photoperiod dependent. In order to describe the impact of grape-seed proanthocyanidins extract (GSPE) on important markers of adipose tissue functionality under an obesogenic environment, we exposed Fischer 344 rats to three different photoperiods and fed them a cafeteria diet for five weeks. Afterwards, we supplemented them with 25 mg GSPE/kg/day for four weeks. Our results revealed that GSPE supplementation prevented excessive body weight gain under a long photoperiod, which could be explained by increased lipolysis in the adipose tissue. Moreover, cholesterol and non-esterified fatty acids (NEFAs) serum concentrations were restored by GSPE under standard photoperiod. GSPE consumption slightly helped combat the obesity-induced hypertrophy in adipocytes, and adiponectin mRNA levels were upregulated under all photoperiods. Overall, the administration of GSPE helped reduce the impact of obesity in the adipose tissue, depending on the photoperiod at which GSPE was consumed and on the type of adipose depots. Obesity is a public health issue worldwide that has nearly tripled since 1975, and in 2016, 39% of adults were overweight and 13% were obese . The WorA healthy functionality of the white adipose tissue (WAT) is crucial to prevent metabolic disorders since alterations in WAT metabolism can lead to an inflammatory response and systemic complications . In an oThe brown adipose tissue (BAT) appears as an important thermogenic tissue in mammals that strongly contributes to energy metabolism. Its role and characteristics are different from WAT: briefly, brown adipocytes contain high amounts of mitochondria and low amounts of small lipid droplets . MoreoveThe consumption of specific bioactive compounds, such as (poly)phenols, is highly associated with health benefits . These mGSPE was kindly provided by Les Dérivés Résiniques et Terpéniques . According to the manufacturer, the GSPE composition used in this study contained monomers 21.3%), dimers (17.4%), trimers (16.3%), tetramers (13.3%), and oligomers  of proanthocyanidins. The exact phenolic composition of GSPE was determined by HPLC-MS/MS  .3%, dime, and cann = 72) were purchased from Charles River Laboratories  and pair-housed in animal quarters at 22 °C with a light/dark period of 12 h. Animals had an adaptation period of 1 week fed with standard diets (STD)  and tap water ad libitum. After the adaptation period, animals were submitted to three different light schedules for 9 weeks to mimic seasonal day lengths: L12 photoperiod (12 h light–12 h darkness); L18 photoperiod (18 h light–6 h darkness); and L6 photoperiod (6 h light–18 h darkness). Animals in each photoperiod group were further divided into three more groups (n = 8); one of them was fed an STD diet ad libitum, and the other two were fed cafeteria (CAF) diet ad libitum. The CAF diet consisted of biscuits with pâté, biscuits with cheese, ensaïmada (sweetened pastry), bacon, carrots, and sweetened milk (20% sucrose w/v) in addition to the standard diet . The CAF diet is a highly palatable diet that is able to induce voluntary hyperphagia. Its composition was 10% protein, 31.9% fat, and 58.1% carbohydrates. Each component of the CAF diet was freshly provided to the animals daily, and they could choose and eat ad libitum. At week 6, the 4-week treatment period started while the animals kept eating the same type of diet. The three STD diet groups received the vehicle (VH) treatment, consisting of an oral dose of water and sweetened milk. Three of the CAF groups (one from each photoperiod) received VH, and the other three received 25 mg/kg/day of GSPE diluted in water and sweetened milk. At the end of the experiment, animals were fasted for 3 h and then sacrificed by live decapitation. Total blood was collected from the neck and then centrifuged  to obtain serum. All adipose tissue depots were excised, weighted and immediately frozen into liquid nitrogen, one piece of epididymal white adipose tissue (eWAT) and inguinal white adipose tissue (iWAT) were also kept in formol for histological analysis. Both serum and tissues were stored at −80 °C until further use. A schematic representation of the experimental design can be found in Eight-week-old male Fischer 344 rats  were measured with enzymatic colorimetric kits . Serum non-esterified fatty acids (NEFAs) were analyzed with the enzymatic colorimetric HR NEFA series kit .2 or >3000 μm2; then, the total number of counted adipocytes to calculate the percentage of adipocytes in both categories.Frozen iWAT and eWAT samples were thawed and fixed in 4% formaldehyde. Tissues underwent successive dehydration and paraffin infiltration immersion  and the paraffin blocks were cut into 2-μm-thick sections using a microtome . The sections were subjected to automated hematoxylin–eosin staining . Sections were observed and acquired at ×10 magnification using AxioVision ZeissImaging software . The area of adipocytes was measured using the Adiposoft open-source software . Four fields per sample were measured, and six samples per group were analyzed. The adipocyte area was calculated from the average value of the area of cells in all measured fields for each sample. The total adipocyte volume was calculated using the formula cyclophilin peptidylprolyl isomerase A (Ppia) mRNA levels and normalized to the levels measured in the corresponding control group. The ∆∆Ct method was used and corrected for primer efficiency [Total RNA from iWAT, eWAT and BAT was extracted using TRIzol reagent  following the manufacturer’s protocol. RNA was quantified in NanoDrop ND-1000 spectrophotometer . The integrity of the RNA was evaluated by the RNA integrity number (RIN) trough 2100 Bioanalyzer Instrument (Agilent Technologies). A RIN higher than six was accepted for total RNA samples. cDNA was synthesized using the High-Capacity cDNA Reverse Transcription Kit  in a Multigene ThermalCycler . The cDNA was subjected to a quantitative reverse transcriptase polymerase chain reaction amplification using iTaq™Universal SYBR Green Supermix  in the 7900HT Fast Real-Time PCR System (Applied Biosystems). The primers used for the different genes are described in ficiency .post hoc test for multiple comparisons. In addition, for biometric, biochemical, and serum variables, a one-way ANOVA followed by Tukey’s post hoc test was conducted to detect significant differences between groups in the same photoperiod. GraphPad Prism 9  was used for all statistical analysis. The values are expressed as means ± SEM. p< 0.05 was considered significant.The effects of both CAF diet administration and seasonal GSPE supplementation on biometric, biochemical, and serum variables as well as gene expression were evaluated by a two-way ANOVA with Tukey’s After nine weeks of study, animals fed the CAF diet significantly increased their body weight, body weight gain, and food intake . HoweverA diet effect was also observed in all circulating metabolic parameters, where CAF consumption significantly increased serum concentrations of glucose, cholesterol, TG, and NEFAs. However, GSPE supplementation was able to restore cholesterol and NEFAs levels in a photoperiod-dependent manner.The histological analysis of iWAT showed a significant effect of the CAF diet on both area and volume of adipocytes in iWAT, which were increased in obese animals . ConsequCebpα and Pparγ  and of Cd36 phenols on health and their natural presence in vegetable foods make these molecules of high interest in research. Additionally, a (poly)phenol-rich diet or the development of natural-based extracts as an alternative for preventing or treating metabolic disorders is more likely to be accepted by society than synthetic options . In thisBifidobacterium and Coprobacillus, while decreased Klebsiella to levels associated with reduced body weight gain and obesity [After nine weeks of study, body weight gain was reduced in response to proanthocyanidin consumption in CAF-fed animals. These results were particularly marked when GSPE was consumed at L18 than when it was at L12 and L6 photoperiods. According to the seasonal adaptations to photoperiod, increased weight gain in long photoperiods appears as a natural metabolic modulation in mammals, which, contrarily, lose more weight in short photoperiods . Neverth obesity . Moreove obesity ,37. In fIl6 and Tnfα), no effects were observed in response to GSPE consumption.Given that WAT functionality responds to changes in photoperiod and that obesity drives major changes to the structure and activity of this tissue, histological analyses of WAT depots were performed. As expected, we observed a strong impact of the CAF diet on the area and volume of adipocytes, which were larger compared to animals fed a chow diet. These changes are common in obesity and contribute to the altered functionality of the WAT. In fact, under an obesogenic environment, the WAT needs to store the excess calories, which can be done through two processes: hyperplasia or hypertrophy . The hypPparγ and Cebpα in both iWAT and eWAT showed an interaction effect between GSPE administration and photoperiod. In iWAT, we observed that GSPE increased its gene expression only in the L12 and L18 groups. Similar effects were previously observed in response to cyanidin-3-glucoside administration [Fabp4 and Cd36) were also affected by GSPE consumption in iWAT (L12 and L18 groups) and in eWAT , which could suggest a beneficial role for proanthocyanidins in helping release circulating TG. In fact, animals consuming GSPE at L12 and L18 photoperiods showed reduced serum TG concentrations compared to VH groups. However, another interaction effect between GSPE and photoperiod on the expression of lipolysis-related genes was detected in both iWAT and eWAT, which were upregulated in response to GSPE consumption only in the L12 and L18 groups. This activation of lipolysis could be involved in reducing the size of existing adipocytes, contributing to β-oxidation and global energy homeostasis. Moreover, the reduction in body weight gain observed in L18 animals could be partly explained by the increased lipolysis detected in these animals. In fact, supplementation with blueberry (poly)phenols suggested increased lipolysis in the WAT [One of the main roles of WAT is to regulate energy balance in the organism. Therefore, lipid turnover is constantly active, involving distinct metabolic pathways that control fat storage and release from the WAT. The process that drives the formation of new adipocytes is named adipogenesis and is mainly driven by the master adipogenic molecules PPARγ and C/EBPs . In our stration  and coul the WAT , and the the WAT .Pparγ, Cebpα), lipid transport , lipolysis , and adipokines , GSPE was capable of returning the expression of these genes to a healthy situation. In this context, (poly)phenol-induced reduction of lipid turnover has been reported previously in human and animal studies [Adipoq. High adiponectin serum concentrations are associated with anti-inflammatory effects that help prevent the metabolic consequences of an obesogenic environment [Following these interaction effects between GSPE consumption and photoperiod, we observed that when proanthocyanidins were consumed at L6 photoperiod, the obesity-driven changes on the gene expression of iWAT were reverted. Thus, while the CAF diet induced the mRNA expression of genes related to adipogenesis phenols consumption. However, other molecules involved in BAT thermogenesis were affected by GSPE in a photoperiod-dependent manner. Thus, proanthocyanidins upregulated the expression of Pgc1α, Cpt1β and Pparα in animals exposed to L12 and L6, while these genes were downregulated in L18 animals. Our results agree with previous findings [Pgc1α expression. However, in our study, we demonstrated that these effects are limited to L6 and L12 photoperiods.Importantly, we also detected a browning effect in iWAT only in animals consuming proanthocyanidins under short photoperiods. In fact, GSPE was associated with increased thermogenesis in the adipose tissue of obese mice , but thefindings , where GIn conclusion, our results indicate that the beneficial effects associated with GSPE consumption on diet-induced obese animals are strongly influenced by the photoperiod of exposure in a tissue-specific manner. Particularly, our findings suggest that L18 photoperiod is highly sensitive to GSPE with respect to body weight adaptation and modulation of metabolic genes, especially adiponectin, in the iWAT. Meanwhile, animals under the L12 and L6 photoperiods showed reduced food intake in response to GSPE and enhanced BAT activity. Therefore, our study reinforces the relevance of considering seasonal rhythms when investigating the metabolic effects of (poly)phenols in the organism in order to properly potentiate the metabolic response of the adipose tissue.


The tumor necrosis factor (TNF) family plays a role in modulating cellular functions that regulate cellular differentiation, survival, apoptosis, and especially cellular immune functions. The TNF family members also play important roles in oncogenesis and progression. However, the potential role of the TNF family members in lung adenocarcinoma (LUAD) is yet to be explored.TNFRGs) in 1,093 LUAD samples was investigated using The Cancer Genome Atlas and Gene Expression Omnibus datasets. The characteristic patterns of TNFRGs in LUAD were systematically probed and three distinct molecular subtypes were identified. Furthermore, a correlation was found between the different subtypes and their clinical characteristics. A TNF scoring system was created to predict overall survival (OS) and therapeutic responses in patients with LUAD. Subsequently, the predictive accuracy of the score was verified and a nomogram was used to optimize the clinical applicability range of the TNF score.The expression of TNF-related genes (TNFRGs analysis of patients with LUAD revealed that TNF could be involved in forming the diverse and complex tumor microenvironment, its clinicopathological features, and its prognosis.A high TNF score, involving the immune and stromal scores, indicated negative odds of OS. Moreover, the TNF score was associated with immune checkpoints and chemotherapeutic drug sensitivity. Collectively, our comprehensive TNFRGs in LUAD, pave a new path for assessing the disease prognosis, and assist in developing personalized therapeutic strategies for patients with LUAD.A TNF-related prognostic model was constructed, and a TNF score was developed. These findings are expected to improve our knowledge regarding the function of  Globally, the incidence of lung cancer has increased and it is the primary cause of cancer deaths worldwide . Non-smaThe tumor necrosis factor (TNF) family contributes to the modulation of cellular functions involved in cellular differentiation, survival, proliferation, apoptosis, and especially immune functions against cancer cells . A studyIn our study, three TNF subtypes associated with clinical consequences were established. Among the three TNF subtypes, based on the differentially expressed genes (DEGs), two gene subtypes exhibiting distinct clinical prognoses and immune cell infiltrations were identified. A scoring system to predict OS was established and used for describing the immune level of LUAD. This system may enhance the understanding of TNF in immune infiltrations and assisted in finding a new direction for more effective therapeutic strategies for LUAD.GSE31210 and GSE72094 were downloaded from the Gene Expression Omnibus (GEO) database. The “Combat” algorithm was used to eliminate batch effects, and three cohorts were combined. Patients with insufficient clinicopathological or survival information were excluded from the study. Our study included 1,093 patients and their detailed clinical information is presented in TNFRGs) were included in the study obtained by reviewing previous studies and sorting out the above mRNA sequencing data. The details of these TNFRGs are provided in TNFRGs. Consensus clustering is a common research method for cancer subtype classification. Samples can be divided into several subtypes according to different sets of omics data, so as to find new disease subtypes or compare and analyze different subtypes. The distribution of the subtypes was confirmed as per the expression profiles of the genes using principal component analysis (PCA).Through “ConsensusClusterPlus”  package DEGs were screened using “limma”  package p <0.05 was considered to be statistically significant. The training and test sets were randomly generated from all the patients with LUAD in a ratio of 1:1. Then, the training set was applied to establish the TNF-related prognostic signature. LASSO Cox regression analysis was used to identify the key genes and corresponding coefficients for model building. The risk score of each patient was calculated based on the standardized expression level of the key genes and their corresponding regression coefficient. The formula was established as follows: Score = GREM1 × 0.077 + GJB2 × 0.068 + CCR2 × −0.151 + MMP1 × 0.002 + IL7R × −0.006 + MS4A1 × −0.062 + HLA − DQB2 × −0.042. Patients were classified into low- and high-risk groups based on the median value of the risk score. The OS among the different groups of patients with LUAD was compared using “Survival” software package.Univariate Cox regression analysis was performed for selecting genes with prognosis value; 50) values of drugs for treating LUAD to explore the differences in drug sensitivity among the patients with different scores.The tumor mutational burden of the TCGA cohort was visualized using “maftools”  package The package “rms” was utilized to construct a nomogram, providing valuable clinical predictive information regarding the clinical characteristics and risk score of patients with LUAD, particularly on 1-, 3-, and 5-year OS. In the nomogram, each clinical variable was mapped with a score and the total score was calculated by adding the scores across all the variables. Calibration plots were used to assess the predictive value among the predicted 1-, 3-, and 5-year OS and the virtually observed outcomes.Using “estimate” package, the stromal, immune, and ESTIMATE scores of each sample were computed using the ESTIMATE algorithm. The abundance of infiltrated immune cells in each sample was assessed using single-sample Gene Set Enrichment Analysis (ssGSEA).p<0.05 was considered statistically significant.All statistical analyses were performed using R 4.1.2 version. The OS was compared between the different subgroups using Kaplan–Meier analysis. Time-dependent receiver operating characteristic (ROC) curve analysis was applied to assess the predictive value of the TNF score. TNFRGs involved in LUAD were observed; 84 of 561 (14.97%) LUAD samples exhibited genetic mutations. The top five mutations were FASLG, TNFRSF8, CD40LG, EDA2R, and EDAR  curve, k = 3 was considered the optimum choice for sorting the entire cohort into subtype clusters A, B, and C . The difference in TNF scores among age, gender, and T grade groups was also analyzed. The TNF scores in those aged <65 years, male and stage T3–4 group were significantly higher than in those aged ≥65 years, female, and stage T1–2, respectively  and immune scores (p < 2.2e-16). However, the stromal score of the high and low TNF score groups was almost the same (p = 0.14). Altogether, the TNF score exhibited a close association with TME in LUAD. Additionally, the correlations between the immune checkpoints and our risk model were investigated in detail. The results revealed that the two risk groups exhibited notably distinct immune checkpoint expressions, such as BTLA, PDCD1, CD274, CTLA4, and CD47 , Natural Science Foundation of Tianjin , Tianjin Key Medical Discipline  Construction Project, Tianjin Health Science, and Technology Project (ZC20179).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.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.


Occupational exposure to dust has been recognised as a significant health hazard to mine workers. This study aimed to investigate the association between exposure to inhalable (INH) and respirable (RES) dust and respiratory health among mine workers in Western Australia using an industry-wide exposure database.The database comprised cross-sectional surveys conducted by mining companies for the period 2001–2012. The study population consisted of 12,797 workers who were monitored for exposure to INH and RES dust and undertook health assessments including a respiratory questionnaire and spirometry test.Despite the general trend of declining exposure to both INH and RES dust observed over the 12 years period, mine workers reported a higher prevalence of phlegm and cough when exposed to elevated concentrations of INH and RES dust. Logistic regression analysis further confirmed the positive association between INH dust exposure and the prevalence of phlegm with an adjusted odds ratio of 1.033 (95% CI 1.012–1.052). Overall, 6.3% of miners might have potential airway obstruction, and exposure to INH dust was associated with impaired lung function parameters.Exposure levels of INH and RES dust particles among mine workers have reduced considerably and were well below currently legislated occupational exposure limits. However, given the reported higher prevalence of phlegm and cough among those with elevated dust concentrations, there is a continued need for effective dust exposure monitoring and control in the mineral mining industry. Mining is commonly defined as the extraction of valuable minerals and other materials from the earth. This is considered one of the major economic activities that contribute to the advancement of economies worldwide. With the current advancement in technology and technological products, the demands for minerals are increasing and consequently the need for mining activities to meet these demands Abraham .However, mine site activities involve significant hazards and risks associated with exposures due to different work processes and the generation or use of hazardous substances ILO . These iMine workers are exposed to dust because the whole process of extracting minerals involves rock breaking through blasting and drilling as well as loading and unloading materials  Regulations The Occupational Safety and Health Administration has a permissible exposure limit for RES dust not to exceed 5 mg/mes NIOSH . The AmeIn the literature, the morbidity and mortality from exposure to dust among mine workers have been well described. However, there is still a lack of any contemporary scientific evidence regarding INH and RES particles from mine inspections over a long period. The objective of the present study was to summarise the recent trend of exposure to INH and RES dust and the associated respiratory health among mine workers by using 12 years of compliance sampling data collected in Australian mineral mines.Mine sites in the State of Western Australia are required to electronically submit their atmospheric contaminant results to the State’s Department of Mines and Petroleum (DMP) to assess compliance with dust regulations. The requested number of samples by DMP is determined on the mine site. For practical reasons, miners with similar exposure profiles are grouped by the mining companies before samples were randomly selected from each similar exposure group. A ventilation officer or hygienist was appointed by each mining company to collate the required data, called CONTAM. The type of mines included in the present study consisted of gold, nickel, iron and other mineral mines.The present study used the 2001–2012 dataset provided by DMP. During the period 2001–2012, a total of 61,482 Western Australian mine workers were monitored for exposures to INH and RES dust. Of this population, 12,797 distinct workers undertook health assessments which constituted our study sample of 13,170 observations as some workers were surveyed more than once during the 12 years. The study population was involved in 129 mining-related activities. To facilitate analysis, these activities were classified into three occupation groups, namely ‘managers’, ‘surface production and services’ and ‘underground mining’. The first group represented mine management occupations, including managers, operation supervisors, superintendents and engineers. The second group included occupations with limited exposure to dust-generating activities, such as geologists, mobile plant operators, electricians and mechanics. The third group comprised those directly involved in underground mining production activities, such as drilling, blasting and loading. The available dataset was summarised by calendar year, occupation, age, gender, work shift length and current smoking status (in the last three months).Workplace atmospheres – Method for sampling and gravimetric determination of respirable dust)   workers were monitored for both RES and INH dust. Air sampling of RES dust was performed using SKC Plastic Cyclone that holds a collection filter in a reusable cassette according to the Australian Standard (dust) AS 2009. Perdust) AS 2009.Mine workers were invited to take part in a health assessment. The health surveillance comprised a respiratory questionnaire, lung function test, an audiometric (hearing) test and in some cases, a chest X-ray. In this study, we were provided with data collected through a respiratory questionnaire and spirometry test which comprised the MineHealth database. The survey consisted of questions related to current respiratory symptoms experienced in the last three months, namely cough, phlegm, wheeze and breathlessness, and further details on the health data collection have been described previously  and health data  which was then entered into the Safety Regulations System (SRS). The CONTAM and MineHealth database were linked, and records were matched by the DMP using each worker’s unique identifier number.The authors were not involved in the data collection and therefore did not seek ethics approval. The data used in the study were compliance sampling data collected in Western Australian mineral mines requested by DMP.Our analysis was based on estimated current exposures to INH and RES, current respiratory symptoms (last three months) and lung function parameters. INH and RES particle concentrations were presented as geometric mean (GM) with geometric standard deviation (GSD) due to their skewed distributions. These geometric means were stratified by personal characteristics , and according to the presence/absence of respiratory symptoms . The overall prevalence of each respiratory symptom was calculated and tabulated concerning personal characteristics. Wilcoxon sign test and Kruskal–Wallis test were used for comparison of geometric means across categories as appropriate, whereas the Chi-squared test was applied to compare the prevalence of respiratory symptoms between personal characteristic groups.To ascertain the association between RES and INH exposure levels and the prevalence of respiratory symptoms, logistic regression models were fitted, with adjusted odds ratios (OR) and their corresponding 95% confidence intervals (CI) for quantifying the magnitude of the observed association, while accounting for the effects of plausible confounding personal factors particularly smoking status. Finally, multiple linear regression analyses were undertaken to assess the relationship between INH and RES exposures and lung function parameters. All statistical analyses were performed in RStudio for Windows (version 3.2.4) (RStudio Team 2012).The mean age of the workers was 36 (SD 11.1) years. Males comprised 90.8% of the mine workers with nearly half (47.3%) being directly engaged in underground mining activities, followed by surface operations (32.5%) and managerial work (20.1%). A great majority (77.6%) of them worked longer shifts (12 h), while about two-thirds (66.4%) reported as non-smokers.p < 0.001).In total, 8561 RES and 5683 INH dust samples were collected from the 12,797 mine workers with reported respiratory health during the period 2001–2012. Figure 3) and RES (0.26 mg/m3) dust were well below the respective  Regulations 3 and 3 mg/m3, with only 2.9% and 0.9% of the samples, respectively, exceeding the limits. Male workers were exposed to significantly higher INH and RES dust concentrations than their female counterparts. The study also found significantly higher mean RES and INH dust concentrations among mine workers who were current smokers when compared to non-smokers , followed by wheeze (13.3%), phlegm (11.3%) and breathlessness (9.8%). Table 3) compared with their asymptomatic colleagues (0.71 µg/m3) (n = 5093).Exposure levels of INH and RES dust were next compared between miners according to their respiratory symptoms. The results showed that the 590 workers who reported phlegm were exposed to significantly higher GM concentrations of INH dust (0.90 µg/mp < 0.001) increase in the prevalence of all respiratory symptoms. Age was also positively and consistently associated with respiratory symptoms; however, the associations with gender, occupation and shift length were less apparent according to the multivariate models.The logistic regression results, overall and stratified by smoking status, are summarised in Table Mean FEV1, FVC and FEV1/FVC of the sample population were 3.97L (SD 0.78), 4.87L (SD 0.94) and 0.82 (SD 0.07), respectively. Overall, 11.9%, 12.4% and 6.3% of the sample might be considered to have impaired lung function in reference to the respective boundary values for the three parameters, FEV1, FVC and FEV1/FVC, respectively. The results from linear regression analyses, overall and stratified by smoking status, are presented in Table 3. This can be explained by the companies’ effort to comply with the stringent occupational health and safety regulations imposed by the DMP in Western Australia. Our estimated GM concentration of RES (0.26 mg/m3) was the same as that measured from underground gold miners in 2003–2004 in Tanzania  observed at the underground gold mine in South Africa during the period 2015–2016 (Nkrumah and Yaw 3)   (Kizil and Donoghue 3) , which subsequently improved after the enforcement by governments under better technical and organisational circumstances. Indeed, our observed declining trend in dust exposures was consistent with previous reports worldwide  and surface production mine workers (GM 0.76 mg/m3) compared with those measured for underground mine workers (GM 0.64 mg/m3). This unexpected finding is difficult to explain because the authors were not involved in the data collection. It is assumed that the UG workers monitored in this study worked in enclosed environments, such as tracks, excavators and boggers, and were better protected from dust exposure compared to those who worked in open spaces and in office environments, including managers and service workers. Dust particles can accidentally be introduced indoors from shoes, clothes and things that are carried inside  (Apelberg et al. Friedman et al.  reportedIt is well known that high exposure to occupational dust can lead to adverse respiratory health. The importance of measuring different dust fractions in work environments has also been acknowledged for the evaluation of exposure and risk to workers (Wippich et al. This study found no significant difference in the prevalence of respiratory symptoms between miners working 10- and 12-h shift except for wheezing. Again, miners on 12-h shift might move to a less dust-generating activity after working long hours, which lead to their reduced prevalence of wheeze than others on 10-h shift.Moreover, our multivariate analyses confirmed that INH dust exposure was positively associated with the prevalence of phlegm, while being a smoker significantly increased the prevalence of all respiratory symptoms. The results are consistent with findings from studies conducted in Tanzania, Ghana and South Africa (Rusibamayila et al. On the other hand, exposure to RES dust appeared to have little association with respiratory symptoms after accounting for the personal confounding factors. Seixas et al.  concludeThe American Thoracic Society and the European Respiratory Society provided guidelines for spirometry assessment (Ferguson et al. The major strength of this study was the use of a large population database with quantitative exposure measurements on INH and RES dust over an extended period of 12 years. However, the following limitations should be taken into account when interpreting the findings. The ‘healthy worker effect’ is a type of selection bias that typically arises in observational studies of occupational exposures Shah . It is rFinally, spirometry tests have been commonly used for assessing lung function among mine workers (Lin et al. This study demonstrated that mine workers still reported a higher prevalence of phlegm and cough when exposed to elevated concentrations of INH and RES dust. The findings underscore the continued need for effective dust exposure monitoring and control for all miners. In addition, it is recommended that mining companies should conduct awareness campaigns on the adverse effects of cigarette smoking on respiratory health.