Rana luteiventris). R. luteiventris occupies naturally fragmented aquatic habitats in this xeric region, but beaver removal, climate change and other factors have put many of these populations at high risk of extirpation without management intervention. We addressed three objectives: (i) assessing support for ESUs within Nevada; (ii) evaluating and revising, if warranted, the current delineation of MUs; and (iii) evaluating genetic diversity, effective population size, adaptive differentiation and functional connectivity to inform ongoing management actions. We found little support for ESUs within Nevada but did identify potential revisions to MUs based on unique landscape drivers of connectivity that distinguish these desert populations from those in the northern portion of the species range. Effective sizes were uniformly small, with low genetic diversity and weak signatures of adaptive differentiation. Our findings suggest that management actions, including translocations and genetic rescue, might be warranted. Our study illustrates how a carefully planned genetic study, designed to address priority management goals that include CU delineation, can provide multiple insights to inform conservation action.Delineating conservation units  is critical to the recovery of declining species because CUs inform both listing status and management actions. Genomic data have strengths and limitations in informing CU delineation and related management questions in natural systems. We illustrate the value of using genomic data in combination with landscape, dispersal and occupancy data to inform CU delineation in Nevada populations of the Great Basin Distinct Population Segment of the Columbia spotted frog ( Conservation unit is a general term for an array of biological and legal categories used to define subspecific population units for the protection and management of intraspecific diversity. Because inappropriate delineation of CUs can waste conservation dollars, hamper recovery efforts and result in inappropriate management actions , which represents major components of intraspecific variability based on genetic, phenotypic, ecological, geographical and life history characteristics. There are many definitions and sets of criteria for defining ESUs  and “significance” , similar to the Waples . DespiteGenetic data can play an important role in informing the complexities of CU delineation and subsequent management and policy decision‐making. Neutral genetic markers, including mitochondrial DNA, microsatellites and single nucleotide polymorphisms (SNPs), can be used to evaluate reproductive isolation in ESUs and, to a limited extent, demographic independence in MUs . R. luteiventris occupies a large range that has been divided into three major clades , the nearby but isolated Ruby Mountains and the geographically disjunct Toiyabe , disease, drought and climate change places many of these populations at high risk of extirpation without management intervention  first identified the Great Basin clade of 5 USFWS, , 2015a. 5 USFWS, . Despites USFWS, . To infoR. luteiventris in Nevada, we addressed three primary research questions: (i) Are there ESUs within Nevada populations of the currently defined Great Basin DPS? (ii) Do genetic data and connectivity analyses support the current delineation of MUs in Nevada? If not, how should MUs be revised to better support management goals? (iii) How should MUs, sites and movement corridors be managed and prioritized for conservation actions such as genetic rescue and reintroduction efforts?To inform ongoing management of 22.1Toe and tail clips were collected between 2010 and 2019 from 31 sites  amplification, degenerate barcodes on the P2 adapter to allow for removal of PCR duplicates  to develop genome‐wide SNPs , distance between stacks (‐M) and distance between catalog loci (‐n). We used 19 individuals (each with a replicate sample) from 16 sites, covering the geographical range of sampling, and tested 13 combinations of parameters starting from default settings . We evaluated mean coverage, number of assembled loci, number of polymorphic loci shared across 80% of individuals, number of SNPs  using the following parameters: ‐m 3, ‐M 2, ‐d, ‐‐model‐type bounded, ‐‐bound‐high 0.05, ‐n 3, ‐‐model marukilow. We applied a coarse filter using populations: ‐p 1, ‐R 0.3, ‐‐min_mac 2. We discarded four individuals with high missingness (>55%) before filtering with the r package radiator  from Hardy–Weinberg proportions in five or more sites . We removed 1328 SNPs that were heterozygous in all individuals  in at least one site . We removed 476 SNPs associated with a weak lane effect in the first library by identifying the contributing SNPs using redundancy analysis (RDA) from the package vegan version 2.5‐6  = 10, 100 and 1000, with 25 repetitions, 200 iterations and 5% cross‐entropy withholding . The RDA used the imputed data set as the response and a dummy variable for library as the predictor. Using a threshold of ±4 SD from the mean loading on the constrained axis, we identified the loci contributing to the lane effect and removed them , biologically relevant environmental predictors derived from the 800‐m PRISM climate normal  at the site level .We assessed reproductive isolation by evaluating population structure using neutral markers (genetic data with candidate adaptive SNPs removed). We calculated pairwise 2.4n = 24) and unsampled but occupied sites . A total of 15,990 adults (>45‐mm snout–vent length) were marked using passive integrated transponder (PIT) tags injected into the dorsal subcutaneous tissue during annual, multipass surveys of all wetlands over a 26‐year period .We used multiple analyses to evaluate MUs including population structure analyses as above. We evaluated functional connectivity in the Jarbidge‐Independence among genetic sites  for sites with sample sizes ≥11 using the LD method of neestimator version 2.01 , characteristics of the sampled sites that could produce migrants contributing to gene flow  and characteristics of the landscape matrix restricting or facilitating gene flow between sites  as our measure of gene flow, calculated from the neutral SNPs using adegenet version 2.1.3 ; therefore, we used the native resolution value (30 m) in all subsequent analyses.We investigated landscape factors influencing functional connectivity in the Jarbidge‐Independence using gravity models .We tested 11 hypotheses of functional connectivity Table . We calc2 Evans, , and perigraph version 1.2.6; Csardi & Nepusz, r| < 0.70.To measure graph‐based connectivity  is included in all hypotheses. We tested pruned networks at 50 km , 100 km maximum distance and saturated  between observations. Because model results were similar, we present 50‐km results as they are more biologically relevant. We used the corrected Akaike information criterion (AICc) for comparison of models fit using maximum likelihood , we predicted the final model  to all wetlands with at least one documented observation between 2001 and 2020 (n = 194). To collect covariate data included in the final model, we used the methods above. We then predicted flow at the population level to a 15‐km maximum distance graph and a saturated  graph. We back‐transformed the natural log flow predictions using the Naihua correction , WILU and UMAG (n = 11), POCR (n = 8), RITI (n = 7) and SFJA (n = 6). The correlation between HO and missingness was low (r = 0.087). Heterozygote miscall rates were low , with 227 candidates most strongly correlated with winter minimum temperature. There were signatures of adaptive divergence across Nevada, with Salmon Falls sites showing differentiation related to high summer precipitation, and Marys River sites related to low winter temperatures. Figure . WICR inFST values, which was very high between the Ruby Mountains and all other sites (mean pairwise FST = 0.66) and the Toiyabe and all other sites  sites. Differences across values of K probably relate to differing levels of past (or possibly contemporary) gene flow. These include assignment of the Owyhee sites TRCR and RITI as either admixed or distinguished as separate groups, splitting of North Fork sites into different groups, identification of DOGE as a separate group, and the separation of Maggie and Marys sites from larger watershed groupings. Variation in assignments among Toiyabe sites are highly variable  across all occupied sites (n = 194) using both a 15‐km maximum distance and a saturated graph . In both cases, we visualized flow  based on the flow prediction (0.852) for the sites with the lowest pairwise FST in the Jarbidge‐Independence region . Flow predictions indicate that functional connectivity is limited across most of the Jarbidge‐Independence, especially peripheral areas. For example, the Salmon Falls, Willow and Maggie Creek sites show no (15 km) to limited (saturated) flow outside their watersheds at the above cutoffs, though admixture results indicate signatures of past connectivity with neighbouring sites. By contrast, flow predictions in the core part of the region show functional connectivity across watershed boundaries, supporting inference from admixture and PCA plots. In particular, TEGU (Bruneau) and NFHU (North Fork) show signatures of gene flow with Owyhee sites.The best‐supported model of functional connectivity was the temperature–moisture model  were among the smallest estimates. Neutral diversity varied widely  and does not include all potential drivers of adaptive differentiation , genetic diversity in these regions is extremely low, indicating that genetic drift is probably the dominant evolutionary force, potentially overpowering selection. While this does not necessarily preclude local adaptation, the current evidence indicates that these regions do not meet the threshold for adaptive differentiation that substantially contributes to the evolutionary legacy of the species.R. luteiventris throughout the historical Nevada range, one of the goals of the 2015 Conservation Agreement and Strategy plan. Importantly, because these regions do not represent areas of significant adaptive divergence based on these genomic results, management actions such as genetic rescue will remain viable options to increase genetic diversity in these isolated areas.However, even if these regions do not meet the threshold of ESU status, their disjunct locations at the southern range boundary are “significant to the species as a whole because…[their] loss would result in a substantial modification of the species' range” , a challenging prospect when funding is limited and/or there are many occupied sites distributed across complex landscapes. In the case of Nevada R. luteiventris, either approach would have been unrealistic to implement given the complex spatial structure among populations and variation in population sizes (clear violations of population genetic models), and the large number of occupied sites (n = 194).Management units are generally defined based on demographic independence . While this approach does not provide a quantitative evaluation of demographic independence, our evaluation of functional connectivity was sufficient to meet the management goals for the species, which are not highly dependent on an assessment of demographic independence . Similarly, the North Fork showed clear within‐watershed diversity, supporting an east–west split based on finer‐scale watershed delineations. Finally, the remainder of the large Bruneau River watershed MU was not assigned due to lack of data; in particular, the southern Bruneau sites have potential functional connectivity with Marys River, but additional genotyping would be needed to verify those projections.Based on this integration of population structure and functional connectivity results, we suggest revision of the MU delineation for Nevada s Figure . First, FST between the Big Smoky Valley site (FAPO) and Reese River sites is slightly higher (0.097) than the average within‐Reese River pairwise FST (0.071), the values are relatively low, especially considering that FAPO is at the southern range boundary. Additionally, PCA and admixture results do not clearly distinguish the FAPO site from others in the Toiyabe, despite little evidence for movement of marked frogs between the Big Smoky Valley and Reese River . These results indicate that the Toiyabe region could be managed as a single MU, especially when efforts are focused on improving genetic diversity across the region (next section).Clearly, the Ruby and Toiyabe regions represent separate MUs, due to their geographical and hydrological isolation. However, genomic analyses do not support the division of the Toiyabe region into two MUs. While the average pairwise 4.3R. luteiventris populations  of the California Channel Islands ranges from 0.00012 to 0.00082 , while two sites in the Reese River had higher estimates (point estimates of 44.7 and 49.0). Increasing Ne at some locations probably reflects the efficacy of ongoing habitat enhancement efforts in the Toiyabe that began in 2004, including drought mitigation efforts  and the use of grazing exclosures   were among the lowest found in sexually reproducing animals. For comparison, nucleotide diversity in endangered island foxes  and/or geographical proximity would be helpful, even if those populations have reduced genetic variability. However, where maximizing genetic rescue and fitness effects is most important, selecting sites with higher diversity  would be best, especially because risks of outbreeding depression should be low  will continue to hamper the utility of genomic assessments of evolutionary potential in at‐risk species (Funk et al., B.R.F. and W.C.F. designed the research in consultation with C.M. and J.P. C.M., J.P., R.V.H. and J.H. collected and coordinated the collection of tissue samples; D.S.P. provided capture–recapture data. B.R.F. and M.M. performed the research and analysed the data. B.R.F. wrote the paper; all authors edited and provided feedback on subsequent versions.The authors declare that they have no conflict of interest.Benefits from this research accrue from the sharing of our data and results on public databases as described above, contributing to the conservation of biodiversity.Figures S1–S10Table S1–S5Click here for additional data file.


Basturma, Smoked Liban,Aushari cheese, Turshi, and Sour Khobz, and 3 fermented beverages, i.e. Shanina, Sharbet Zbeeb, and Erk Sous in Iraq, are documented. Traditional fermented foods and beverages have multiple health benefits because of high content of probiotics and bioactive compounds. Traditional fermented foods and beverages are made using the back-slopping technique which ensures safety of production and maintains organoleptic properties. The review highlights the potential of fermented foods and beverages for their large-scale commercialization.The tradition of making fermented foods and beverages in Iraq dates back to 7500 BC. These fermented foods and beverages are represented by meat-, milk-, vegetable-, and fruit-based products reflecting diversity of agricultural production in ancient Iraq (Mesopotamia). Although the recipes for some fermented foods and beverages were lost throughout history, those remaining foods and beverages occupy a noticeable position in modern Iraqi cuisine. In this review, knowledge and techniques for preparation of 5 traditional fermented foods, i.e.  Ga-har” [sikura” [Iraq is historically known as the Mesopotamia, the region formed between the rivers of Tigris and Euphrates, often referred to as a cradle of ancient civilizations . Due to Ga-har” . The Sum[sikura” . The Sum[sikura” .Fermented foods and beverages were integral to many ancient diets due to pleasant taste compared to the raw materials of which these fermented foods and beverages were made. Fermented foods represented an important source of nutrients in a time of crisis due to their extended shelf life. For example, the study of the most recent crisis in Iraq confirmed the importance of traditional fermented foods in sustaining nutrition of Iraqi households in critical times . The preTurshi, fermented mixed vegetables, including cucumbers, bell pepper, cabbage, and garlic, is an important element of modern Iraqi cuisine [Basturma, seasonal fermented lamb minced meat, is a popular winter foodstuff in Iraq that is consumed because of high protein and energy content [Shanina (yogurt beverage), is consumed in Iraq daily and Sherbet Zabeeb (fermented raisin) is consumed during the Ramadan.Traditional fermented foods and beverages are part of multiple cultures, and some of these foods and beverages have been produced on a large scale. For example,  cuisine . Basturm content . SimilarLactobacillus, Bifidobacterium, Streptococcus, Leuconostoc, Pediococcus, Propionibacterium, Bacillus, and Enterococcus) and fungi  [γ-aminobutyric acid (GABA), fatty acids, amino acids, and vitamins [Although fermented foods and beverages have traditionally been consumed because of their pleasant taste, the biotechnology analysis demonstrates significant health benefits associated with consumption of traditional fermented foods and beverages. These benefits are associated with the presence of microorganisms known as probiotics, including bacteria (Candida) . In addivitamins .Fermentation involves two techniques, i.e. spontaneous fermentation and back-slopping fermentation. Spontaneous fermentation is performed by mixing the raw material with brine, and fermentation is done by several microorganisms associated with this raw material. Back-slopping fermentation is carried out by mixing the ingredients with subsequent sterilization required to deactivate the microorganisms associated with the raw material. Following this, a small amount of previously fermented ingredients is added to the sterilized ingredients and further fermented at room temperature. According to Li and Gänzle , back-slFermented foods and beverages have been popular with consumers who consider these foods and beverages important for the immune system, especially during COVID-19 . TraditiFertile Crescent region in northern Iraq [Iraq (Mesopotamia) is a region in the Middle East with hot and dry summers  and moderate winters  which is due to its climate ranging from the Mediterranean to hot semi-arid continental . Iraq isern Iraq . In the ern Iraq . The ricBasturma, Turshi, Sharbet Zbeeb, and Erk Sous), Erbil (Smoked Liban), Sulaymaniyah (Aushari cheese), Baghdad ), Babil (Turshi Medabis), and Al Muthana (Turshi Medabis). Traditional fermented foods and beverages were first studied through the literature review; this desk-based research was then supplemented with field observations and interviews administered with local small-scale producers and households. A total of 11 small-scale producers (third to fifth generation) and 8 households (women aged 58–64) were interviewed in this study. The interviews documented traditional preparation methods, mode, and time of consumption and examined the sociocultural significance of traditional foods and beverages in Iraq. Information about the current opportunities and challenges of making and distributing traditional fermented foods and beverages was collected from small-scale producers.This ethnographic research is based on traditional knowledge of fermented foods and beverages that was transferred in Iraq from generation to generation. Primary data were collected following the method described by Fontefrancesco , with soPhoenix dactylifera L) fruits. The ancient city of Nineveh, a former capital of the Assyrian Empire, is known for fermented foods prepared from vegetables, wheat, and barley. In the southern city of Basrah located in the vicinity of the Arabian Gulf, traditional fermented foods are prepared from fish, such as mackerel (Scomber scombrus) and cobia (Rachycentron canadum). The preparation techniques are another source of diversity of Iraqi fermented foods as they show significant cross-regional differences. This section will highlight the preparation methods and the raw materials applied to the reviewed traditional fermented foods in Iraq.Traditional fermented foods in Iraq are regionally diverse due to the rich history of the country. For example, in the ancient city of Babylon, fermented foods are derived from date  is traditional fermented milk produced via the lacto-fermentation process using well defined starter cultures. The history of yogurt is not well known, but scholarly evidence indicates that yogurt has originated in the Mesopotamia at around 5000 BC. [Liban; it is consumed with bread daily as a main dish for breakfast [Liban in Iraq that are traditionally prepared via back-slopping fermentation and made using different types of milks including cow’s, sheep’s, and goat’s milk. Traditional smoked Liban is unique for Iraq, and the most authentic product can be found in the north of Iraq, i.e. in the city of Erbil. Smoked Liban is well known among the Iraqi as the Liban Erbil due to its high quality and unique flavour.5000 BC. . In severeakfast . There aLiban is prepared using sheep’s milk, i.e. milk of higher cost and quality compared to cow’s milk. According to this study’s informants, sheep’s milk is preferred by the locals due to its aroma. The Liban prepared from sheep’s milk has high physiochemical qualities and is rich in flavours because of high content of protein, solids, lipids, and polyunsaturated fatty acids [Liban are due to a unique processing technique practiced by local producers  3–4 times to develop the dominant starter cultures of preferred flavours. The technique was observed to improve quality and refine safety of dairy products by establishing the dominant starter cultures with antimicrobial and probiotic properties [Liban is consumed by the local people in Iraq daily and mostly in the morning during breakfast.There are two traditional methods to develop the unique flavour for smoked operties . HoweverAushari is traditional semi-hard cheese made of sheep’s and goat’s milk in the mountains of northern Iraq [Aushari is mainly produced by the Kurdish tribes and can be found in the Kurdistan region, especially in Sulaymaniyah and Erbil. The villagers collect sheep’s or goat’s milk early in the morning, and milk is heated for 10–15 min and then cooled down for 30–45 min. This sterilized milk is used to prepare soft cheese using rennet enzymes from the fourth stomach of calves (abomasum). The curd is pressed using muslin clothes to remove the whey and reduce moisture content. The curd is shaped as cheese balls weighing 300–400 g each. The pressed soft cheese balls are placed in bags made of sheep stomach that have been pre-cleaned using water and salt. A traditional herb known locally as the Jaje belonging to the Thyme (Thymus capitatus) family is added to the bags to enhance flavour of Aushari. The bags are placed in sheepskin and transferred to the caves at the top of the mountains that have high humidity and low temperature. The cheese is matured for 6–8 months. During maturation, the cheese develops special aroma and taste. The ripening process is due to rennet enzymes, i.e. the enzymes from the sheep stomach, the enzymes from herbs, and natural microflora.ern Iraq . AushariAushari cheese has yellowish colour and a very strong, “sharp” aroma which is due to prolonged aging and added herbs , cauliflower (Brassica oleracea var. botrytis), cabbage (Brassica oleracea var. oleracea), green olive (Olea europaea L.), Jerusalem artichoke (Helianthus tuberosus L.), bell pepper (Capsicum annuum), and garlic (Allium sativum) [Turshi Medabis and the method known as the Turshi Mahshi from the historical city of Nineveh. Turshi is prepared via back-slopping fermentation using a portion of the previous batch that is added to the new batch. Turshi Medabis has a sweet–sour taste due to the mixture of flavouring ingredients including date syrup, apple cider, and tamarind.sativum) . There aA traditional recipe for preparing 10 kg of the finished product involves 3 kg cucumber, 2 kg cucumber, 2 kg cauliflower, 1 kg green olive, 1 kg Jerusalem artichoke, and 1 kg green chilli. Vegetables are washed and transferred to fermentation tanks to be covered with brine . The first spontaneous fermentation is carried out for 30–40 days. The product is then kept in water for 24 h to remove impurities and extra salt. The fermented vegetables are subjected to second fermentation with water and date syrup (25% v/v) and kept at room temperature for 5–15 days.Turshi Mahshi is prepared following the first fermentation process of Turshi Medabis using cucumbers and bell pepper only. In the second stage of fermentation, vegetables are stuffed with coriander, parsley, garlic, celery, and chilli. The stuffed vegetables are placed in a tank and fermented for 3–5 days with water containing 5% lemon juice and 10% date vinegar. The traditional Iraqi Turshi is served with a special condiment known as the Amba that is made by mixing pickled green mangoes, white vinegar, turmeric, fenugreek, and salt  and Falafal (patties made of ground chickpeas).alt Fig. D. Amba iTurshi is commonly consumed in Iraq as an appetizer during lunch and dinner, with consumption frequency increasing in the winter. Due to low acidity provided by lemon and vinegar, the product has long shelf life (10 days at room temperature). However, acidity can reduce quality and preferred texture of fermented vegetables. Therefore, Turshi is recommended to be consumed within 2–3 days. Burris et al. [Salmonella enterica. Cauliflower, cabbage, and bell pepper are also prone to outbreaks due to potential contamination with Listeria [Turshi has been reported responsible for an outbreak of food-borne Botulism caused by Clostridium botulinum among the immigrants to the USA [Turshi in Iraq. However, no outbreaks have been recorded due to consumption of Turshi for a long time. This can be a result of strict hygienic conditions maintained in local production facilities.s et al.  report hListeria . Recentl the USA . There aKhobz is traditional sour bread in Iraq made of whole wheat flour. The art of this bread’s production has been practiced for centuries by ancient civilizations in southern Iraq, such as the Babylon, Sumerian, Uruk, Lagash, and Ur. These ancient civilizations utilized date fruits as a starter culture for dough preparation because date fruits were believed to have exceptional health benefits [Khobz. Importantly, bread made using date fruits or syrup can be found in several Arab countries including Bahrain where it is known as the Khubez Tamer [Sour benefits . Date frbenefits . Currentez Tamer . Additioez Tamer .The traditional production method is simple and begins by mixing whole wheat flour (1 kg) with 1 cup of water 250 mL) to prepare the dough. The starter culture is prepared by mixing ripen date fruits (10–15 pieces) with 250 ml water which is kept at room temperature overnight. The starter culture is added to the dough at the recommended ratio of 1:5 w/w to ensure good fermentation. The dough is kept overnight for leavening and baked in the oven at 180–220 °C to produce thick sour bread  (1 kg) that are soaked in water (2 L) and kept being fermented spontaneously for 16–24 h with mint leaves (100 g). This sludge-resembling mixture is extracted using an industrial juice extractor and filtered using fine mesh bags. Filtered juice is then mixed with water at the ratio of 1:1 v/v and sugar (240 g). This fermented juice is placed in dispenser machines and subsequently consumed as a fresh cold beverage  via spontaneous fermentation. The beverage is consumed in the summer as a refreshment drink and in the winter as an energy drink. Popularity of Erk Sous in the Iraqis’ culture is due to the abundance of liquorice plant at the water rims of the Tigris and Euphrates. Liquorice roots were found on clay tablets from the Babylon civilization and in the Assyrian Empire [n Empire . Liquorin Empire .Erk Sous, liquorice roots are mixed with water at the ratio of 1:1 and subjected to milling to form a paste using mortar and pestle. The paste is then kept in a pottery jar in darkness for 2 days. The paste is soaked in water overnight, and the resultant juice is filtered using muslin cloth  reduces the potential of traditional foods and beverages to spread outside Iraq. For example, manufacturers of soft drinks, such as Coca-Cola and PepsiCo, have established their presence across the world and their products replace traditional beverages due to aggressive marketing. However, growing consumer demand for healthy foods and beverages can drive commercialization of traditional fermented foods and beverages which are healthier than many globally known brands . This deHowever, in developed countries, fermented foods and beverages are not commonly consumed and/or commercialized at an industrial scale . DespiteThe restrictions to import fermented foods by the Food and Drug Administration of the USA and the European Food Safety Authority of the European Union have limited the spread of fermented foods and beverages in developed countries. Further, most fermented foods and beverages produced in developing countries suffer from ineffective packaging with resultant short shelf life. This complicates the logistics of fermented foods and beverages from production to consumption. However, several fermented foods and beverages have been exported from developing countries to developed countries and become highly accepted and consumed such as Kimchi  and TempTraditional fermented foods and beverages are derived from various ancient civilizations, and their preparation techniques have been inherited over centuries from one generation to another. Although traditional fermented foods and beverages of Iraq originate from ancient diets, they remain popular nowadays among the local people. However, no research has attempted to document these traditional fermented foods and beverages and explore their production techniques. No study has been undertaken to assess the risk of traditional fermented foods and beverages in Iraq to public health and their association with outbreaks. This is a major shortcoming as traditional fermented foods and beverages of Iraq have significant commercialization potential commercialization given their health benefits and pleasant taste.This current study has reviewed traditional fermented foods and beverages of Iraq. By employing the method of ethnographic research, the study has documented the main production techniques. By supplementing findings with the literature review, the study has also shed light on safety of traditional foods and beverages in Iraq. Lastly, the study has briefly discussed the potential for traditional Iraqi foods and beverages to upscale their production.In terms of future research outlook, studies should aim at identifying the acceptable levels of food safety and hygiene required to prepare traditional foods and beverages and establish the data on their microbiology loads. Moreover, the implementation of the HACCP system will significantly improve safety of fermented foods and beverages of Iraq. Research should also be carried out to profile bioactive compounds and evaluate health benefits of traditional fermented foods and beverages. Studies are also necessitated to determine how traditional fermented foods and beverages of Iraq can effectively compete with modern foods and beverages, especially from the perspective of their perceived healthiness and marketing and particularly in light of COVID-19. Consumption habits of different generations, particularly Generation Y and Z, should be investigated given that these consumer groups are characterized by higher health concerns. A cost benefit analysis of producing traditional foods and beverages in Iraq should also be conducted to calculate the expense of their prospective commercialization.


Aegilops tauschii Coss. as a donor of wheat D genome has an important role in wheat breeding programs. Genetic and phylogeographic diversity of 79 Ae. tauschii accessions collected from north and northwest of Iran were analyzed based on retroelement insertional polymorphisms using inter-retrotransposon amplified polymorphism (IRAP) and retrotransposon-microsatellite amplified polymorphism (REMAP) markers.In total, 306 and 151 polymorphic bands were amplified in IRAP and REMAP analyses, respectively. As a result, a high level of polymorphism was observed among the studied accessions as revealed by an average of 25.5 bands per primer/primer combination and mean PIC value of 0.47 in IRAP and an average of 25.16 bands per primer combination and mean PIC value of 0.47 in REMAP. Genetic relationships of the accessions were analyzed using distance- and model-based cluster analyses.Ae. tauschii in Iran should provide important knowledge on genetic resources and their applications in wheat breeding programs.The result showed that genetic distance did not seem to be related to geographic distribution, and the accessions could be divided into three groups, which was further supported by principal coordinate analysis. These results on genetic diversity and population structure of The online version contains supplementary material available at 10.1186/s43141-022-00363-y. T. turgidum; 2n = 4× = 28, AABB) and Aegilops tauschii  in the Fertile Crescent resulted in hexaploid bread wheat  [Ae. tauschii Coss. (2n = 2× = 14) as a donor of wheat D genome is a source of many favorable genes for important agronomic traits [Ae. tauschii, D genome of wheat has the slightest diversity [Ae. tauschii with the widest geographical distribution and greater genetic variation compared with the corresponding homologous loci in the D genome of bread wheat is a promising genetic resource for broadening wheat genetic background. Considering the wide genetic variation in Ae. tauschii germplasm and its easy crossability with wheat, Ae. tauschii accessions have been exploited by various groups throughout the world for wheat improvement [Approximately, 8000 years ago, a spontaneous hybridization between cultivated emmer wheat ( AABBDD) . Ae. tauc traits , resistac traits , stripe c traits , grain yc traits , and cadc traits . Among tiversity . Thereforovement –15.tauschii with extended cylindrical spikelets and ssp. strangulata with moniliform spikes bearing quadrate spikelets have been identified in Ae. tauschii. Three distinct varieties, namely var. anathera, var. meyeri, and var. typica, have been recognized within ssp. tauschii, while ssp. strangulata contains only the var. strangulata. The description of Ae. tauschii variants followed the morphological characteristics revealed wide geographical distribution for ssp. tauschii around Eurasian continent, while ssp. strangulata has a confined dispersal [tauschii is spread across the entire Ae. tauschii geographical range, whereas ssp. strangulata is only present in Transcaucasia and around the Caspian Sea region of Iran. However, recently, one ssp. strangulata accession is reported from Uzbekistan, which is the first report for this subspecies out of their natural habitat [T. aestivum and Ae. tauschii showed a closer fit with ssp. strangulata than ssp. tauschii. According to earlier genetic investigations, Transcaucasia and southwestern Caspian Iran are the origins of bread wheat [Ae. tauschii and wheat accessions using the Ae. tauschii 10 K Infinium single-nucleotide polymorphism (SNP) array and identified 12 Ae. tauschii accessions, each closely related to a wheat D genome chromosome. All 12 accessions belonged to spp. strangulate collected from southwestern and southern Caspian Sea, Iran. Genetic diversity of Ae. tauschii’s germplasm has been extensively studied using various molecular techniques such as allozyme [Based on spike morphology, two subspecies including ssp. ispersal . The ssp habitat . The anaad wheat , 19. Wanad wheat  analyzedallozyme , RFLP [1allozyme –25, IRAPallozyme , SNPs [3allozyme , 28, AFLallozyme , and genallozyme .T. urartu and T. boeoticum from west and northwest of Iran by IRAP and REMAP markers. Grouping of the accessions using REMAP data could differentiate them according to their species and geographical origin, but grouping based on IRAP could not separate the accessions of two species. However, based on both marker systems, considerable diversity was observed among and within the studied populations.Cereal genomes consisted of an extraordinary number of transposable elements, in particular, LTR retrotransposons, which is highly dynamic. It is reported that the LTR retrotransposons are often found in different densities or copy numbers among individuals of the same species –33. TherAe. tauschii’s genome contains at least 66% LTR [Ae. tauschii accessions. Few studies have been utilized retrotransposon-based markers to evaluate genetic variability in collections of Ae. tauschii. Saeidi et al. [Ae. tauschii from north and center of Iran, using IRAP markers, and reported high levels of genetic diversity in the studied accessions. However, IRAP data could not differentiate the accessions based on their geographical origins. Boyko et al. [Ae. tauschii. 66% LTR ; therefoi et al.  analyzedo et al.  mapped 8Ae. tauschii accessions from north and northwest of Iran.In the present study, we used IRAP and REMAP markers to analyze genetic diversity and population structure of a newly collected Aegilops tauschii Coss. were collected from north and northwest of Iran. For each accession, 10–15 plants at least 2 m apart were randomly sampled. The name and geographical information of collection sites were described in the Seventy-nine accessions of Sukkula, Nikkita, and BARE-1 families of barley (Hordeum vulgare L.) genome including Sukkula, Nikkita, LTR6150, LTR6149, 5′LTR1, 5′LTR2, and 3′LTR  non-denature polyacrylamide gel and detected by ethidium bromide staining using a Gel-Scan 3000 electrophoresis system .Seven LTR retrotransposon primers from e) [The IRAP and REMAP amplification profiles were scored as the presence (1) or absence (0) at each polymorphic band position for each primer/primer combination in all accessions. Each polymorphic band was treated as a single locus with two alleles. Monographic bands were not scored and used in the analyses. The polymorphism information content (PIC), Shannonʼs information index, and Nei’s gene diversity (He)  were cale) .Ae. tauschii accessions was evaluated by the neighbor-joining algorithm (NJ), for which the trees were constructed using MEGA 7.0 software [Ae. tauschii accessions was performed based on IRAP and REMAP by using the Bayesian Markov chain Monte Carlo model-based clustering implemented in the software package STRUCTURE v2.3.4 [Ae. tauschii accessions. The Nei’ gene diversity (He), Shannon’s information index (I), and within-population variation (WP) were calculated for each geographical population. The analysis of molecular variance (AMOVA) was performed using GenAlex 6.5 [Aegilops tauschii geographical populations.Pairwise, genetic distance between accessions was calculated based on the minimum evolution evolutionary distance coefficient  using IRsoftware . SupportE v2.3.4 . The modE v2.3.4 , using oE v2.3.4 . The accE v2.3.4  to furthAlex 6.5  to calcuAe. tauschii accessions generated using 5′LTR primer are shown in Fig. Banding patterns of IRAP amplicons for some The parameters relating to the genetic diversity of geographical groups including the number of polymorphic loci (NPL), Nei’s gene diversity (He), Shannon’s information index (I), and within-population diversity (WP) are shown in Table Ae. tauschii accessions generated using Sukkula and ISSR6 primers combination. Out of 18 LTR and ISSR primer combinations examined in the REMAP analyses, six primer combinations produced scorable bans. A total of 151 polymorphic bands with an average of 25.16 markers per primer combination were amplified. The highest (44) and lowest (13) number of polymorphic markers were amplified using ISSR6 + 5′LTR1 and ISSR42 + Sukkula combinations, respectively. The PIC value varied from 0.42 (ISSR42 + Sukkula) to 0.49 (ISSR6 + Nikkita and ISSR6 + Sukkula) with an average of 0.47. Based on all polymorphic primer combinations, Mazandaran and Golestan with 88 and 81 loci had the highest and lowest number of polymorphic loci, respectively. The maximum and minimum average number of polymorphic loci were also scored in Mazandaran and Golestan populations with values of 14.67 and 13.50, respectively  and the Mazandaran 0.27) populations, respectively, and Shannon’s index ranged from 0.56 (Guilan) to 0.39 (Mazandaran). The highest level of within-population diversity was observed in the Golestan population (29.18), and the Mazandaran population revealed the lowest within-population diversity (23.40)  and lowest (0.064) distances were found between “Azerbaijan and Mazandaran” and “Golestan and Guilan,” respectively, which was in agreement with their geographical distances. Among the populations, the Azerbaijan population was far genetically relative to others. In REMAP analysis, the highest genetic distance (0.213) was also observed between Azerbaijan and Mazandaran populations and the lowest (0.043) between Golestan and Guilan populations.Analysis of molecular variance (AMOVA) revealed high genetic variation within populations and low genetic differentiation among populations. The proportion of variation attributable to within-population differences was high, 90, 94, and 92% by IRAP, REMAP, and combined data, respectively.Ae. tauschii accessions were assessed using the minimum evolution distance coefficient based on IRAP, REMAP, and combined data. Mantel test revealed low and nonsignificant correlation between two matrices . The resulting distance matrices were used to construct dendrograms using the neighbor-joining clustering algorithm. The model-based hierarchical structure among the studied accessions was also conducted by STRUCTURE v2.3.4 software [The genetic relationships among 79 software  using alK = 2  was plotted versus ΔK, which presented a clear peak at K = 6  with a mean PIC value of 0.25 was produced [In the present study, the genetic diversity and population structure of 79 r et al.  used 15 i et al.  detectedproduced .barley for fingerprinting and biodiversity studies [Sukkula, Nikkita, and BARE-1 families of barley to analyze genetic relationships among Ae. tauschii accessions. Five out of the seven examined primers and seven primer combinations allowed the amplification of multiple and distinguishable fragments in the 79 Ae. tauschii accessions, and the degree of polymorphism was very high, 100% by either method. In addition to the detection of high IRAP and REMAP polymorphism among the 79 accessions, polymorphism within each geographical population was also high for most of the primers/primer combinations , lineage 2 (L2), possible hybrids, wheat, and Ae. tauschii collection combined and reported the highest Nei’s diversity index for L2 = 0.1326 followed by L1 = 0.0872 and wheat of 0.0158. Higher values of the Nei’s index indicate greater allelic diversity in a given population.The values of intrapopulation genetic diversity based on Nei’s gene diversity using IRAP data ranged from 0.24 among accessions from the Azerbaijan population to 0.48 among accessions from the Guilan population. Based on REMAP data, it ranged from 0.27 for Mazandaran’s accessions to 0.38 for Guilan’s accessions. The same trends within-population genetic diversity were observed based on Shannon’s information index . It could be due to different genomic regions amplified by these two marker systems. Using the IRAP, genomic regions are amplified by two nearby retrotransposons using outward-facing primers, whereas, in REMAP, amplification between retrotransposons proximal to simple sequence repeats (microsatellites) produces the marker bands .Mantel test revealed a low and nonsignificant correlation between the two matrices (Ae. tauschii, 75 hexaploid wheats, and seven tetraploid wheats conclude that southwestern Caspian Iran is the center of wheat genetic diversity and the center of origin of bread wheat. Therefore, studying Ae. tauschii accessions from the wheat center of origin using molecular markers has an essential role in wheat breeding programs. We analyze the genetic diversity of 79 accessions mainly collected from the Caspian area using two highly polymorphic markers: IRAP and REMAP. The results of the molecular genetic diversity analysis clearly showed that this collection of Ae. tauschii will be helpful for wheat breeding programs as revealed by high PIC and diversity indices and the number of polymorphic markers.Wang et al.  used 781Additional file 1: Supplementary table. Name and geographical coordination of the studied Ae. tauschii accessions.


Research on the origin and domestication of Bactrian camel is helpful to understand the process of animal evolution and plays an important role in protecting the genetic diversity of Bactrian camel populations.New advances in research of the origin and domestication of Bactrian camel have been acquired by the rapid development of next-generation sequencing technology, molecular genetics, and bioinformatics.An overview given about the distribution, breeds, evolutionary history, and domestication, as well as the genetic diversity based on mtDNA, nuclear, and whole-genome sequencing of Bactrian camel populations.Camelus bactrianus) adapt to the specific weather conditions of both desert and semi-desert regions and provide a wide range of useful products to Gobi Desert rural communities, including meat, milk, and wool. In animal taxonomy, the Bactrian camel belongs to Animalia, Chordata, Mammalia, Placentalia, Artiodactyla, Tylopoda, and Camelidae. The family of Camelidae consists of the tribes Camelini (Old World camels) and Lamini (New World camels). The Camelini includes the Camelus genus, and the Lamini is divided into two genera, Lama and Vicugna. The genus Camelus includes three species, domesticated single-humped camel , distributed in the arid deserts of North Africa, East Africa, and the Arabian Peninsula; domesticated two-humped camel , found in cooler areas of Asia (Camelus ferus), considered critically endangered by the International Union for Conservation of Nature (Lama glama) and alpaca (Vicugna pacos) and two wild species of Guanaco (Lama guanicoe) and vicuña (Vicugna vicugna)  . The claTo survive and live in harsh conditions, camels developed many special abilities and attributes. They can adapt to desert and semi-desert conditions—cold, hot, arid, poor grazing, and even food shortages, and can withstand extreme thirst and hunger for a long time due to sufficient energy in their humps and abdomen . The bodIn this review, we aim to provide a comprehensive summary of the distribution, breeds, evolutionary history, and domestication, as well as the genetic diversity based on mitochondrial DNA (mtDNA), nuclear, and whole-genome sequencing of Bactrian camel populations.Bactrian camel numbers and distribution vary from region to region. The ecological geographical distribution is in the transition from grasslands to desert areas; the higher the degree of desertification, the more Bactrian camels there are. Although the service value of Bactrian camels has substantially decreased, the trend of nurturing camels in the desert is increasing at the global level. Of the entire Bactrian camel population, more than 90% are found in China, Mongolia, and Kazakhstan. The Bactrian camel population was estimated to be 381,000 in China, 401,000 in Mongolia, and 170,000 in Kazakhstan in 2016 according to the Food and Agriculture Organization (FAO) statistics. The growth trend of Bactrian camels in China, Mongolia, and Kazakhstan from 2006 to 2016 is shown in Based on distribution areas and phenotypic characteristics, several different breeds of Bactrian camel exist. In China, Bactrian camels are mainly distributed across Inner Mongolia, Xinjiang, Qinghai, and Gansu and are classified into five breeds: Alxa, Sonid, Qinghai, Tarim, and Junggar Bactrian camel . MongoliIn addition, wild two-humped camels were discovered by Nikolaj Przewalski in 1878 . As earlProtylopus in the North American savannah during the Eocene period (~45 million years ago), measuring about 80 cm long and weighing 2.6 kg, with no hump, resembling the current hare . During this period, the Earth environment began to become dry and cold, and grasslands were replaced by sand. With the change in the environment, the Procamelus evolved a unique tooth and jaw structure similar to modern camels and had a similar pace as modern camels. During the Pliocene period (2.5 million years ago), North America and South America were connected, which brought opportunities for the migration of the Paracamelus, and the progenitors of Lamini entered South America around 3 million years ago  by using the “BABA/ABBA,” the study demonstrated that it will not influence the results by removing the introgressed genomic segments  has the characteristics of simple structure, maternal inheritance, a high evolution rate, and no recombination and has been used as a genetic marker of genetic diversity in livestock. The first molecular evolutionary analysis of the family Camelidae described by the mitochondrial cytochrome b (Cytb) gene  revealedIn different domestic breeds, researchers analyzed the genetic diversity and population structure based on the different mitochondrial segments. The genetic diversity and phylogeographic structure were detected based on the 809-bp mtDNA fragment  and the Recently, a comprehensive study was completed to reveal genetic diversity by mtDNA control region sequences of 182 individuals from Old World camels . Thirty-In Mongolian domestic breeds, researchers traced possible differentiation within and between the three local breeds  and the common Mongolian native camel (MNT) . The nucP < 0.001) with 9.6% of the total genetic variance present among the populations and the remaining 90.4% among individuals within the populations (A previous study focused on the genetic diversity of Bactrian camels from China and Mongolia. The population genetic diversity was studied on 254 Bactrian and 5 wild two-humped camels from China and Mongolia using genotype data from 16 microsatellite markers . The anaulations . Chuluunulations .−3) was significantly higher than that of Bactrian camels from all geographic regions (0.88 × 10−3 to 1.11 × 10−3); among the Bactrian camels, the wild two-humped camels showed the lowest π (0.88 × 10−3) compared with all the domestic populations, consistent with the previous nucleotide diversity estimates based on mitochondrial genomes (−3 to 1.11 × 10−3) than those living in East Asia (0.95 × 10−3 to 1.02 × 10−3). The tendency was also confirmed by Watterson’s θ (Similar to its late domestication history, the Bactrian camel is one of the last livestock animals to have its population genome sequenced and genetic diversity revealed. The domestic Bactrian camels were chosen to cover as many major geographic regions as possible, including China, Mongolia, Kazakhstan, Russia, and Iran and compared to the dromedary from Iran . The pai genomes , 2017. Irson’s θ .The Bactrian camel is a key species in the Gobi Desert, and it is also a unique large livestock animal living in desert and semi-desert environments around the world. Because most of the distribution areas of Bactrian camels are areas where ethnic minorities gather, Bactrian camels have been closely linked to ethnic minorities for thousands of years, playing an irreplaceable role in the reproduction and survival of ethnic minorities. The Bactrian camel has a special status among mammals domesticated by humans since they are highly adapted to the extreme desert ecosystem. It is a multipurpose animal used for dairy production, racing, and transportation. China, Mongolia, Russia, and Kazakhstan are the countries with the largest populations of Bactrian camels in the world. It is imperative to comprehensively and systematically study the genetic diversity, phylogeny, population differentiation, and inbreeding of Bactrian camels in different regions at the molecular level, so as to provide a scientific basis for the protection of Bactrian camel germplasm resources and variety cultivation.


After the addition of known substrates like unlabeled estradiol-17β-glucuronide, estrone-3-sulfate, bromosulfophthalein, protoporphyrin X, rifampicin, and taurocholate to the outside of the preloaded CHO cells, we observed efflux of [3H]-estradiol-17β-glucuronide due to exchange with the added compounds. Of the tested 30 compounds, some organic cation transporter substrates like diphenhydramine, metformin, and salbutamol did not induce [3H]-estradiol-17β-glucuronide efflux, indicating that the two OATPs do not transport them. However, 22 (for OATP1B1) and 16 (for OATP1B3) of the tested compounds resulted in [3H]-estradiol-17β-glucuronide efflux, suggesting that they are OATP substrates. Among these compounds, we further tested clarithromycin, indomethacin, reserpine, and verapamil and confirmed that they are substrates of the two OATPs. These results demonstrate that the substrate spectrum of the well-characterized organic anion transporting polypeptides includes several organic cations. Furthermore, as for other drug uptake transporters, the CCF assay is an easy-to-use screening tool to identify novel OATP substrates.OATP1B1 and OATP1B3 are two drug transporters that mediate the uptake of multiple endo- and xenobiotics, including many drugs, into human hepatocytes. Numerous inhibitors have been identified, and for some of them, it is not clear whether they are also substrates. Historically radiolabeled substrates or LC-MS/MS methods were needed to test for transported substrates, both of which can be limiting in time and money. However, the competitive counterflow (CCF) assay originally described for OCT2 and, more recently, for OCT1, OATP2B1, and OATP1A2 does not require radiolabeled substrates or LC-MS/MS methods and, as a result, is a more cost-effective approach to identifying substrates of multidrug transporters. We used a CCF assay based on the stimulated efflux of the common model substrate estradiol-17β-glucuronide (E17βG) and tested 30 compounds for OATP1B1- and OATP1B3-mediated transport. Chinese Hamster Ovary (CHO) cells stably expressing OATP1B1 or OATP1B3 were preloaded with 10 nM [ Numerous chemicals have been reported to interact with the two liver-specific OATP1B1 and OATP1B3  and [3H]-verapamil (83.3 Ci/mmol) were purchased from PerkinElmer . [3H]-clarithromycin (12.2 Ci/mmol) and [3H]-reserpine (10 Ci/mmol) were obtained from Moravek Biochemicals . [3H]-indomethacin (5 Ci/mmol) was purchased from American Radiolabeled Chemicals . Unlabeled compounds were purchased from Sigma-Aldrich ; unlabeled compound 6 -2--5-propyloxy-4-oxo-4H-chromene) was described before (Radiolabeled [d before . All othChinese Hamster Ovary (CHO) cells stably expressing OATP1B1*1b or OATP1B3 haplotype 1 were cultured as described before . Uptake 3H]-estradiol-17β-glucuronide in uptake buffer . . . Then efp < 0.05.All calculations and statistical analyses were performed using Prism 9 . Significance was determined using one-way ANOVA followed by Dunnett’s multiple comparisons. Results were considered significantly different at 3H]-estradiol-17β-glucuronide into CHO cells stably expressing OATP1B1 or OATP1B3 on 96-well plates at room temperature over 3 h. As shown in 3H]-estradiol-17β-glucuronide for 2 h, and then efflux was initiated by the addition of 100 μM estradiol-17β-glucuronide (efflux) or an equal volume of water (control). To perform the competitive counterflow assay, steady-state conditions for the respective transporter must first be established. Therefore, we measured the uptake of 10 nM [3H]-estradiol-17β-glucuronide efflux. 3H]-estradiol-17β-glucuronide. The negative controls, glucose, glutamic acid, and ouabain (for OATP1B1), did not result in the efflux of [3H]-estradiol-17β-glucuronide. Glucose even resulted in a slight but significant increase of [3H]-estradiol-17β-glucuronide. For OATP1B3, the results were similar , clarithromycin (p = 0.0626), cimetidine, and gemfibrozil (p = 0.109). Finally, the last nine compounds produced unexpected efflux patterns. Like OATP1B1, gramicidin-mediated efflux was not concentration-dependent and only reached 30—40%. We saw the same pattern also with oleanolic acid. For nifedipine, efflux was more pronounced at 1 μM as compared to 10 μM. Ursolic acid triggered stimulation of uptake for estradiol-17β-glucuronide at 100 μM. Diosmetin, clotrimazole, and gliquidone stimulated uptake at 1 μM. Pararosaniline stimulated uptake at 10 μM and 100 μM. In contrast, compound 6 stimulated uptake at all three concentrations.For OATP1B3, the results are summarized in Based on the results in 3H]-estradiol-17β-glucuronide. Except for pravastatin, the positive and negative controls behaved as expected (see below). We tested 30 compounds and identified 20 novel substrates for OATP1B1  than OATP1B1 (104 μM). In addition, only SNPs in the Solute carrier of the OATP1B1 gene (SLCO1B1) have been associated with changes in pravastatin pharmacokinetics  , and it osporine . Such inBromocriptine is almost entirely metabolized in the liver . In addi3H]-estradiol-17β-glucuronide at each of the three concentrations and for both transporters indicates that it may interfere with the driving force of OATP-mediated transport. In the future, experiments with vesicles from OATP-expressing cells and gramicidin should be performed to elucidate this mechanism better. The increased uptake instead of efflux of [3H]-estradiol-17β-glucuronide in OATP1B3-expressing CHO cells due to nifedipine and gliquidone could indicate that they are substrates of OATP1B3 and inhibit an ABC efflux transporter whose activity is involved in the steady-state loading of [3H]-estradiol-17β-glucuronide. Nifedipine (ABCG2) which is expressed in CHO-K1 cells (3H]-estradiol-17β-glucuronide uptake rather than no effect. We assume that this has something to do with the still unknown transport mechanism of OATP1B-mediated uptake. It is clear that OATP1B1 works as an exchanger and the addition of glucose could have generated more of the normal physiological compound that exchanges for OATP1B1-mediated substrate uptake. Further experiments are required to elucidate this finding in more detail.Regarding compounds that resulted in unexpected efflux patterns, gramicidin, nifedipine, diosmetin, and gliquidone behaved similarly for both OATPs. Gramicidin is an unspecific ionophore that permits the rapid diffusion of potassium, protons, and alkali cations. The fact that it resulted in a consistent efflux of -estradiol-17β-glucuronide as the tracer substrate. We identified several novel substrates for both OATPs, particularly the additional organic cations, clarithromycin, and verapamil. Future screens, e.g., for anticancer drugs, could be performed with this assay to identify which of the many drugs that interact with OATP1B1 and OATP1B3 are transported and pose a risk for potential liver toxicity.In summary, we have used the CCF assay for OATP1B1 and OATP1B3 with [


Oral squamous cell carcinoma (OSCC) usually originates from the precancerous lesions of oral mucosa and accounts for approximately 90% of oral cancers. Current therapeutic approaches do not yet meet the needs of patients, and the 5-year survival rate of patients with OSCC is only 50%. Recent studies have revealed that the signal transducer and activator of transcription 3 (STAT3) plays a key role in the development and progression of OSCC. STAT3 is overexpressed and constitutively activated in OSCC cells, and promotes cancer cell proliferation and anti-apoptosis, migration and invasion, angiogenesis, radiotherapy resistance, and immune escape, as well as stem cell self-renewal and differentiation by regulating the transcription of its downstream target genes. Inhibitors of the STAT3 signaling pathway have shown the promising anticancer effects in vitro and in vivo, and STAT3 is expected to be a molecular target for the treatment of OSCC. In this review, we highlight the oncogenic significance of STAT3 in OSCC with an emphasis on the therapeutic approaches and effective small molecule inhibitors targeting STAT3. Finally, we also propose the potential research directions in the expectation of developing more specific STAT3 inhibitors for OSCC treatment. Oral squamous cell carcinoma (OSCC) represents the most frequent form of head and neck squamous cell carcinoma that is the sixth most common group of cancers worldwide ,2. OSCC The signal transducer and activators of transcription (STAT) family are potential cytoplasmic transcription factors, including STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b, and STAT6, which can be activated in response to cytokine and growth factor stimulation to mediate multiple intracellular signaling pathways . STAT prRecently, researchers have found that STAT3 is overexpressed and constitutively activated in OSCC and plays an important role in OSCC aggressiveness ,21,22. GSTAT3 is tightly regulated by negative modulators to maintain an inactive state in an unstimulated cell . These nIn the classical STAT3 signaling pathway, the p-STAT3 dimer is able to bind to the corresponding site on DNA and initiates nuclear transcription. Indeed, STAT3 is also localized in mitochondria as a monomer. Mitochondrial STAT3 (MitoSTAT3) can regulate complexes I and II to play a role of modulator for mitochondrial respiration ,30. MitoStudies have shown that overactivation of STAT3 in many types of tumors may depend on the following mechanisms: excessive stimulation caused by overexpressed cytokines and growth factors such as interleukin-6 (IL-6), interleukin-10 (IL-10), epidermal growth factor (EGF) as well as fibroblast growth factor (FGF); hyperactivation of receptors for cytokines or growth factors; elevated activity of cytoplasmic non-receptor tyrosine kinases, such as Src and Abl kinase; loss of negative regulation for STAT3 caused by inactivation or decreased expression of endogenous negative regulators, e.g., PIAS, SOCS, and PTPs. These mechanisms can induce uncontrolled cell growth, malignant cell transformation, angiogenesis, metastasis, invasion, and immune escape . ImportaPorphyromonas gingivalis and Fusobacterium nucleatum were responsible for the upregulation of cyclin D1 via IL-6/STAT3 dependent-mechanism to drive the OSCC growth [Several studies have demonstrated that STAT3 promotes cell proliferation and inhibits apoptosis in OSCC by increasing the expression of target genes, including survivin, Mcl-1, c-Myc, Glut5, cyclin D1, B-cell lymphoma-2 (Bcl-2), and B-cell lymphoma extra -large (Bcl-xL) ,57,58,59C growth . MiR-769C growth . FurtherC growth .Epithelial–mesenchymal transition (EMT) is an important biological mechanism underlying the metastasis of primary tumors . During Consistent with the above findings, the inhibition of the JAK2/STAT3 may significantly suppress p-STAT3-induced migration and invasion of OSCC cells. Terminal differentiation-induced non-coding RNA (TINCR) suppressed migration of OSCC by highly reducing the expression of JAK2, p-JAK2, STAT3, and p-STAT3 . AldehydThe pro-angiogenic role of STAT3 has been partially attributed to the upregulation of IL-8, MMP-9, vascular endothelial growth factor (VEGF), angiopoietin 2 (Angpt2), and hypoxia-inducible factor 1-alpha (HIF-1α) via p-STAT3 transactivation ,80,81. RIt has been observed that blocking STAT3 signaling enhances the anticancer activity of chemotherapies in OSCC, thus endorsing a critical role of STAT3 in regulating the chemosensitivity of OSCC . OverexpPrevious work suggested that STAT3 was a powerful regulator of tumor immunosuppression, and activation of the JAK2/STAT3/PD-L1 signaling axis played a crucial role in the immune escape of osteosarcoma and cervical cancer ,89. In ORecently, reports have shown that the JAK2/STAT3 signaling pathway also plays a critical role in the EMT and stemness of OSCC. For example, C-C chemokine receptor 7 (CCR7) and its ligand chemokine ligand 21 (CCL21) were reported to be abnormally abundant in OSCC tissues, and CCR7 expression was correlated with EMT and the stemness of OSCC. The treatment with JAK2 inhibitor AG490 could reduce the promotive effects of CCL21 on OSCC cells colony formation and sphere formation . OverallSTAT3 phosphorylation status is capable of influencing autophagy in OSCC cells. Compound 59, an AMPK activator, inhibited JAK/STAT3 signaling, arrested cells in the G1 phase and promoted autophagy. These findings supported the potential of compound 59 for the treatment of OSCC patients through the suppression of STAT3 pathway . IcaritiRecently, increasingly more studies have shown STAT3 to contribute to radioresistance. Matsuoka et al. found that the IL-6/STAT3 pathway was relevant in resistance to radiation , and oveTME is a highly complex and heterogenous ecosystem consisting of tumor infiltrating cancer cells, immune cells, and other cells. STAT3 is not only activated in cancer cells, but also becomes hyperactivated in immune cells within the OSCC TME. Some studies have suggested that activation of the JAK2/STAT signaling pathway impaired the antitumor activity of immune cells. For example, radiation treatment of head and neck cancer induced STAT3 signaling to enhance the abundance and function of regulatory T (Treg) cells and resistance to radiation therapy. STAT3 inhibition was beneficial in patients receiving radiation therapy . IL-6-inAbundant evidence has suggested that STAT3 may be a promising molecular target for OSCC treatment ,108,109.Various STAT3 inhibitors have been developed and shown some efficacy in OSCC in vitro and in vivo, which are showed in Targeting the intrinsic STAT3 inhibitors, such as SOCS and PTPs, has been considered as a potential strategy for repressing STAT3 signaling pathway. For example, Bovine lactoferrin attenuated the growth of OSCC through increasing SOCS3 activation and then enhanced SOCS3-mediated STAT3 dephosphorylation and inactivation . A microSTAT3 inhibitors have been developed to interact with the STAT3 domains, with a major focus on the SH2 domain and the DBD. For instance, N-4-hydroxyphenylretinamide (4-HPR) was highly bound at STAT3’s dimerization site and c-Abl and c-Src ATP-binding kinase sites to suppress cancer-promoting pathways including STAT3 phosphorylation, STAT3-DNA binding, and production of the trans-signaling enabling sIL-6R . The STAOther STAT3 inhibitors were found to inhibit STAT3 phosphorylation, whereas they have not been investigated for the binding ability with STAT3 and ability to regulate upstream regulators of STAT3. For example, Niclosamide inhibited the migration and invasion in OSCC cells through the downregulation of p-STAT3 at Tyr705 . NitidinThe STAT3 inhibitors we introduced above face many problems in their application. For example, how to target cancer cells or immune cells to improve the efficacy; the low cell permeability also prevents the inhibitors from being fully utilized; and the toxic side effects caused by off-target effects need to be addressed. Specificity and selectivity of these inhibitors of STAT3 have been questioned. Currently, a growing number of preclinical and clinical studies showed that antisense oligonucleotide (ASO)-induced STAT3 silence became a promising approach for lymphoma and lung cancer therapy. However, ASOs targeting STAT3 may cause thrombocytopenia, and toxicity limitations hindered the development of drugs ,112. In As mentioned above, inhibitors targeting STAT3 or its upstream regulators may cause off-target effects, which are connected to the following factors: cell membranal or intracellular signals may deliver in a network, not in a single track; structural conservation of STAT family members; and diversity of STAT3 target genes. These underscore the need to develop inhibitors to regulate downstream targets of STAT3. Precise obstruction of STAT3 downstream signals could help prevent the unintended consequences of general STAT3 inhibition. For example, STAT3 has been reported to act as a transcription factor for Nicotinamide N-methyltransferase (NNMT), and upregulated NNMT in OSCC can contribute to proliferation and invasiveness ,150. NewAlthough some progress has been made in the treatment of OSCC, its prognosis remains poor, with a 5-year survival rate of nearly 50% . A comprInterestingly, contrary to its accepted tumor-promoting role, a fraction of research has shown an opposite role of STAT3 in cancer cells. It was observed that STAT3 knockdown promoted the growth of MDA-MB-231 cell-derived xenograft tumors . AnotherMany STAT3-targeted therapies have been successfully developed and have shown efficacy in preclinical models of OSCC in vitro and in vivo. However, cancers including OSCC are multifaceted heterogeneous diseases . OSCC ha


The current study evaluates genetic heterogeneities in response to trauma among U.S. young adults. Using Add Health Wave III, which coincidently overlapped with the September 11 attacks, and a depression mean and variance polygenic scores, we investigate how the polygenic scores moderate the causal effect of 9/11 on mental health. Our results show the presence of genetic heterogeneity, where those with high genetic plasticity experience an increase in depressive symptoms following trauma while those with low genetic plasticity do not. While the documented differences in reactions to trauma are important, we also note our ability to predict responses based only on genetic measures are too imprecise to identify susceptible clinical patients. We, therefore, contend that the expected benefits from genetic screening to identify susceptible individuals after trauma are limited. Our results provide novel evidence of a specific source of an additional heterogeneity contributing to the inequality of health following trauma. With the rise and availability of genetic data, the hypothesized associations between genotype and depressive symptoms have begun to be examined. immediate response to trauma. Given that most individuals do not develop depressive symptoms at all or eventually recover to the normal level after trauma  targets predicting levels of an outcome, a recent methodological advancement allows us to capture sensitivities of an outcome towards environmental exposures , a “vari1.2.9/11 was a series of terrorist attacks by al-Qaeda on the morning of September 11, 2001. Aircrafts were crashed into the World Trade Center in Manhattan, New York; the Pentagon in Arlington County, Virginia; and Stonycreek Township in Shanksville, Pennsylvania. Approximately 3,000 people died and more than 6,000 people were injured. Adult New Yorkers who repeatedly saw victims falling from the World Trade Center reported a higher prevalence of posttraumatic stress disorder and depressive symptoms than other New York residents . VictimsPrevious studies provide empirical evidence for different responses to 9/11 among New Yorkers. For example, a longitudinal study over 12 years found four different trajectories of post-traumatic stress disorder among the World Trade Center disaster responders: 76.1% of resilience, 7.5% of recovery, 12.1% of delayed, and 4.4% of chronic trajectories . Further1.3.“Should we seek to identify the most susceptible children and disproportionately target them when it comes to investing scarce intervention and service dollars? I believe the answer is yes” .The availability of genetic data in social scientific datasets makes scholars question whether genotypes can be used to target susceptible individuals to increase the efficiency of policy interventions. Despite the growing interests in using genotypes to maximize benefits of policy intervention, there are remaining challenges that scholars should resolve to suggest such genetic screening. Boardman and Fletcher  encourag1.4.Hypothesis 1. Young adults who carry a higher genetic risk of psychological problems are more likely to suffer from psychological disturbance immediately after 9/11 than their counterparts who carry a lower genetic risk of psychological problems.The diatheses-stress model leads us to test for genetic heterogeneities in response to trauma. Nonetheless, no studies have used polygenic score measurements to examine the genetic heterogeneities in immediate response to trauma for young adults. To fill this gap in the existing literature, we test the following hypothesis by using an established quasi-experimental design:Hypothesis 2. Young adults who carry a higher genetic plasticity of psychological problems to environmental shocks are more likely to suffer from psychological disturbance immediately after 9/11 than their counterparts who carry a lower genetic plasticity.Further, we also test the following hypothesis in consideration of a recent methodological advancement of variance polygenic score:By testing these hypotheses, we also aim to provide some assessment of whether benefits of genetic screening to identify susceptible individuals after trauma are plausible.2.2.1.We used data from the National Longitudinal Study of Adolescent to Adult Health  . Add Hea2.2.immediate response to trauma. The RDD assumes no other treatment and exchangeability around cut-points, which implies respondents cannot sort around the threshold.To investigate genetic heterogeneities in response to trauma, we used a sharp regression discontinuity design (RDD) . An RDD 2.3.2.3.1.We used a set of questions from a Center for Epidemiology Studies Depression (CES-D) screener  asking r2.3.2.We employed two polygenic scores  as genetic measurements. A polygenic score is created using the cumulative effects of segregating loci of small effect to phenotypes . A polygOur primary genetic measurements were the mean and variance depression polygenic scores. We used the mean depression polygenic score provided by Add Health. Following the recommendation of Add Health , we usedAs a validity check for the mean depression polygenic score, we evaluated the association between mean depression polygenic score and the CES-D scores after accounting for first 10 principal components to account for population structure-related confounding effects  in our a2.3.3.We used exposure to 9/11 as our measure of trauma. Data of Add Health Wave III were collected over 2001 and 2002, which overlapped with 9/11. The binary treatment variable was constructed from the interview dates. Individuals interviewed after 9/11 were classified into the treatment group (coded as 1), and those interviewed prior to 9/11 were classified into the control group (coded as 0). All respondents interviewed on September 11, 2001, were classified into the treatment group because the terrorist attacks occurred early on the morning of September 11. We also used the interview date as a running variable and centered at September 11, 2001. Because 9/11 consisted of a series of unexpected exogeneous traumatic events, we assumed the unidirectional causation .2.3.4.Adding control variables within an RDD setting was motivated to improve precision of the treatment effects  and accoSome responses to the questions used to construct these control variables were missing, with a missing rate of 0.2% (CES-D score in Wave I) to 32% (baseline extraversion). To avoid losing respondents with missing data, we used multiple imputation via chained equations to impute missing values of these control variables . Ordered2.4.2.4.1.The choice of bandwidths is a major issue in the RDD and shapes the precision and bias of estimates. We used multiple arbitrarily selected bandwidths  to confiTi indicates treatment assignment, ri indicates interview dates, and χi indicates a set of covariates except the first 10 principal components. β1, which is our interest, indicates the local average treatment effect of 9/11 on the CES-D score. (β1 + β3) denotes the slope of interview date after 9/11. We employed a linear equation because an appropriately chosen bandwidth adjusts the accuracy of approximation relative to higher-order polynomial equations  no treatment other than 9/11 and (2) exchangeability around cut-points which implies respondents could not sort around the threshold. First, we conducted placebo regressions for the CES-D score to test the significance of the observed discontinuity at other interview dates (August 22 to October 1) with bandwidths of 20 days prior to and after the cut-point. The RDD also assumes that the treatment and control groups have similar characteristics. To test this exchangeability assumption, we estimated the RDD with a dependent variable of CES-D score and 19 items of the CES-D score in Wave I, neuroticism, extraversion, conscientiousness, age, sex, state of residence where at least 40 respondents in our analytical sample lived within the bandwidths of 50 days, family income, mother’s educational attainment, PVT score, the first 10 principal components, and mean and variance depression polygenic scores with bandwidths of 50 days. Out of 64 tests, the discontinuities of the proportion of living in state 27 , the proportion of mothers with some college degree , family income , and principal component 9  reached a conventional 5% significance level. Most results showed no significant discontinuities and the effect sizes relative to their standard deviation are not as large as that of the CES-D in Wave III. However, the number of significant discontinuities was slightly larger than would be expected by chance. This may be due to postponements of an interview among those living close to the stricken areas. Nonetheless, the Add Health anonymizes the place of residence and does not provide information on interview postponements; therefore, we cannot empirically evaluate that speculation. Instead, we added state of residence as a control variable to account for this possibility. After controlling state of residence, the discontinuity of the proportion of mothers with some college degree was no longer statistically significant. Overall, we found no evidence to question the validity of the RDD in the balance check. Additionally, the non-significant discontinuities of mean and variance depression polygenic scores demonstrate the validity of gene-environment analysis, which assumes no gene-environment correlation.3.3.1.3.2.immediate increase of the CES-D score after 9/11. The slope of interview dates on the CES-D score prior to 9/11 is positive, whereas the CES-D score shows negative association with the interview dates after 9/11. Results are consistent in The visualized output of the RDD with bandwidths of 50 days prior to and after 9/11 is presented in 3.3.We then turned to the RDD with a variance depression polygenic score. 4.Our goal in this study was to evaluate genetic heterogeneities in the immediate response to trauma with a quasi-experimental design. Our first finding, replicating other works, is an increase of the CES-D score immediately after 9/11. We also found that the CES-D score was not associated with the interview dates prior to 9/11 but negatively associated with the interview dates after 9/11—following a recovery trajectory. These results match theoretical predictions because the majority of the sample are expected to recover to the normal level of depressive symptoms even if they experience an increase of depressive symptoms immediately after trauma .We considered genetic heterogeneities in response to trauma by using two depression polygenic scores. Using the mean depression polygenic score, there were no detectable genetic heterogeneities in response to trauma. This is likely because the mean depression polygenic score captures the level of psychological distress, but not the plasticity of mental health to environmental exposures. Nevertheless, our findings are not consistent with the diathesis-stress model  and the By contrast, individuals with a heightened genetic plasticity/sensitivity of mental health to environmental exposures showed a greater vulnerability immediately after trauma than those with a lower genetic plasticity. This finding is particularly important in light of the emerging interest in genetic plasticity in gene-environment research. Consistent with previous research , our resDespite the presence of suggestive visual results for differences in the pace of recovery by the variance depression polygenic score, we did not find statistically significant differences in the pace of recovery by the mean or variance depression polygenic scores. Although bereavements cannot be equated with exposure to 9/11, this is not consistent with the findings that the pace of recovery after a stressful life event is faster among older people with a high mean depression polygenic score than those with a low mean depression polygenic score . This inOur results also provide important implications for policy interventions. We confirmed a larger increase of depressive symptoms after 9/11 among those with a high variance depression polygenic score. This implies the possibility of policy interventions to improve the pace of recovery to achieve the normal level of depressive symptoms for those who experienced a larger increase of depressive symptoms after trauma. Nevertheless, given that the suggested cut-off score for significant depressive symptoms in the 10-item CES-D score is 8 or above , the effOur results provide an empirical basis for genetic heterogeneities in the immediate response to trauma, but the study is limited in several ways. First, the nature of current social genomics research precludes the inclusion of non-European ancestry respondents. Because the accuracy of prediction of polygenic scores relies on training GWAS sample sets  and mostSecond, the observed genetic heterogeneities in response to 9/11 may not be due to genetic differences. Because polygenic scores are constructed for prediction from GWAS, polygenic scores for socially determined outcomes may reflect not only genetic differences but also socio-environmental differences . FurtherThird, there may be a measurement error in the polygenic scores. Indeed, a prior MTAG study suggested the large amount of measurement error in the mean polygenic score . This laFinally, given that the majority of the sample does not develop chronic psychological problems, our approach precludes us from identifying the differences in genetic profiles among resilient, recovery, delayed distress, and chronic distress trajectories. How do genetic profiles among those who experienced the delayed or chronic distress trajectories differ from those who experienced the resilient or recovery trajectories? Studies to address such question would advance the discussion of policy interventions by genotypes for a chronic mental disorder after trauma.Despite these limitations, our study makes valuable contributions to empirical evidence of genetic heterogeneities in response to trauma. We were able to document genetic heterogeneities in response to 9/11 by using a variance depression polygenic score, an emerging genetic marker for gene-environment research. Of particular importance for the current debate in policy interventions is evidence for the limited benefits of genetic screening to efficiently intervene susceptible individuals. We also documented the possibility that policy makers may fail to intervene with victims in need of public support if they rely on genotypes to target susceptible individuals. Although we cannot argue that policy makers should depend on genetic markers to target susceptible individuals, these novel insights provide an additional heterogeneity contributing to the inequality of health. Subsequent extensions of this research may investigate differences in genetic heterogeneities by non-European ancestry groups with more diverse GWAS training datasets, use a larger sample with a stronger statistical power to detect more precise genetic heterogeneities in response to trauma, and examine genetic profiles among the four prototypes of the trajectory of response to trauma. A growing availability of genetic data in nationally representative datasets has been stimulating scholarly interests among social scientists and makes it possible to not only control genetic markers but also develop a novel dimension of heterogeneity. Revisiting accumulated wisdoms in social sciences with genetic data and investigating interactive associations between genotypes and environmental factors are important tasks for social scientists to deepen the understandings of social phenomena.1