In general, jar fermentation with aged brine contributed more flavor to pickled chili peppers than other procedures.The effects of (fresh/aged) brine and (pool/jar) containers on the flavor characteristics of pickled chili peppers were investigated based on a multivariate analysis integrated with kinetics modeling. The results showed that the effect of brine on organic acid, sugar, and aroma was more dominant than that of containers, while free amino acids production was more affected by containers than brines. Chili pepper fermented using aged brine exhibited higher acidity (3.71–3.92) and sugar (7.92–8.51 mg/g) than that using fresh brine . Besides, chili peppers fermented using pool containers showed higher free amino acids content (424.74–478.82 mg/100 g) than using a jar (128.77–242.90 mg/100 g), particularly with aged brine. As for aroma, the number of volatiles in aged brine was higher (88–96) than that in fresh brine (76–80). The contents of the esters, alcohols, and ketones were significantly higher in the aged brine samples than those in fresh brine ( Natural fermentation is an ancient technique widely used for food processing. It can extend shelf life, improve flavor profile, and increase nutritional and functional values ,2. AmongPickle jars are traditionally used as the main fermenting vessels to provide the fermented vegetable with a unique flavor, particularly porcelain jars . Anaerobβ-myrcene, linalool, (E)-linalool oxide, neryl oxide, and α-terpineol) were the main aroma compounds detected in fermented chili peppers [Paocai [Pickled chili pepper is one of the most typical fermented vegetables in southwest China . Our res peppers . During  peppers . In addi [Paocai . The pla [Paocai .Based on these previous studies, it can be deduced that fermentation brines and containers are the main variables that the industry has paid high attention to and wants to control in order to produce high quality products with stable flavor properties. Therefore, it is necessary to systematically study the effects of fermentation brines and containers on the quality of fermentation products, in order to obtain the high-quality fermentation methods in a large-scale industrial production. However, such research is obviously lacking at present. To illustrate this issue, the present study aimed to evaluate the role of different brines (fresh/aged brine) and large-scale containers (pool/jar) on the flavor formation of fermented chili peppers during natural fermentation. Taste-related attributes  and aroma-related attributes (headspace volatile compounds) of chili pepper were investigated during the fermentation. The results of this study could guide the industry to select proper fermentation procedures (brines and containers) for different types of fermented chili peppers production.N-Alkanes  were provided by Anpel .Standards  were purchased from Aladdin . High-performance liquid chromatography (HPLC)-grade acetonitrile and methanol were obtained from Sigma-Aldrich . The standard amino acid solution (type H) was acquired from Wako . Xiaomila (Capsicum frutescens L.) were fermented in pools or jars with fresh brine  or aged brine  at an ambient temperature (20–25 °C) for a month. In the preliminary experiment, the changes in pH and OD600 nm of the fermented chili peppers followed by different fermentation procedures were monitored (data not shown). Based on the results of the preliminary experiment, the sampling time moments were selected as 0, 1, 4, 7, 14, and 30 days during fermentation. For each sampling moment and fermentation procedure, samples (~100 g) were taken at three sampling points from the top to the bottom of the pool/jar, and four sampling points from the side to the center of the pool/jar. The collected samples for each time points (~700 g) were mixed into sterile bags. Sterile bags were used for sampling to reduce the external effects on microorganisms. After sampling, they were quickly frozen using liquid nitrogen and immediately transported to the laboratory for physicochemical parameters and flavor analysis.Fermented chili pepper samples were produced by Yunnan Hongbin Green Food Group Co., Ltd.  under large-scale fermentation procedures including pickle pool with fresh brine (PFB), pickle pool with aged brine (PAB), pickle jar with fresh brine (JFB), and pickle jar with aged brine (JAB). Fresh green The pH value was determined according to the previous study . BrieflyThe extraction and measurement of organic acids in the fermented chili peppers samples were conducted as per our previous study . Brieflyv/v). Glucose, fructose, and sucrose were identified and quantified with their respective standards. Each sample was extracted and analyzed in triplicate.The extraction of sugar in the fermented chili peppers samples was the same as for the organic acids. The extract was analyzed by an HPLC system  and an ELSD detector  equipped with a column . The analysis condition was set at 1 mL/min with an injection volume of 5 μL, and isocratic elution with water and acetonitrile  was equipped with an ion-exchange resin 2622 column  and a UV detector was used at 440 (proline) and 570 (other FAAs) nm. Amino acid mixture (type H) was used as the standard to calculate the FAA content. Each sample was extracted and analyzed in triplicate.FAAs were extracted and analyzed as per our previous study . The homThe volatile compounds of the samples were determined by using gas chromatography-mass spectrometry (GC-MS)  coupled with headspace-solid phase microextraction (HS-SPME), as described in our previous study with some modifications . Brieflym/z 35–500, the electron ionization mode was 70 eV, and the ion source temperature and interface temperature were 230 °C and 280 °C, respectively. Each sample was subjected to three parallel experiments.A GC-MS system fitted with a DB-5 MS column  was used to separate and detect the volatiles. The initial temperature of the GC column oven was kept at 45 °C for 5 min, then increased to 250 °C at a rate of 5 °C/min, maintained for 2 min, and then cooled to the initial temperature. The carrier gas was helium at a flow rate of 0.74 mL/min. The scanning range of the mass spectrometer was n-alkanes  under the same operating conditions. Internal standard (3-octanol) calibration was also conducted for semi-quantification.Tentative identification of volatile compounds was performed based on the comparison of the database from the NIST 2014 library and the experimentally determined retention index (RI), which was calculated using Data were presented as mean value ± standard deviation. Results were analyzed using one-way analysis of variance (ANOVA) and the Origin 2021 software . Tukey’s test using IBM SPSS  was performed at a significance level of 0.05.X variables, and brines and containers were considered as categorical Y variables. Variable identification (VID) coefficients were calculated to select discriminant aroma compounds, and those with absolute VID values above 0.800 were selected [The clustered heatmap was illustrated for organic acids and FAAs data by TBtools . The influence of brines and containers on flavor compounds was analyzed using a partial least squares discriminant analysis (PSL-DA) model on Solo . The flavor compounds were considered as selected .C is the pH at fermentation time (days), C0 is the initial value on day 0 of fermentation, ∞C is the value of the stable fraction, k is the reaction rate constant, and t is the number of days of fermentation.Fractional conversion kinetic modeling was carried out by using Equation (1) in Origin  to fit the pH changes of 4 fermented chili peppers with the fermentation time.p < 0.05). The pH of fermented chili peppers significantly decreased within 7 days (p < 0.05) and then maintained a stable value during fermentation. After 30 days, the pH values of fermented chili peppers varied from 3.71 and 3.96  and fell within the ranges of 3.2–4.2, indicating that the peppers in this study were considered to be fully fermented and ready to eat [The pH value is the main parameter for determining the fermentation stages of pickled vegetables . As showy to eat .R2 > 0.90), and the change rates of JFB (k = 0.62) and JAB (k = 0.69) were higher than that of PFB (k = 0.39) and PAB (k = 0.41) (2) [2 leads to an acidification cultivation broth, which leads to an increase in the pH of the fermented chili peppers [Under the same fermentation container, the pH value of chili peppers fermented with aged brine was lower than that fermented with fresh brine A. The in = 0.41) A. This m.41) (2) . An incr peppers .1 included most of the aged brine group fermented during 4–30 days, category B2 included the fresh brine group fermented during 4–30 days, and category B3 included all chili pepper samples fermented during 0–1 days. It indicated that the effects of fermentation brines were dominant in that of containers on the organic acid profile. In addition, there were large gaps in the organic acid profile between pickled chili peppers fermented before and after 4 days (p < 0.05)  were detected in fermented chili peppers. The changes in organic acid content during fermentation of four kinds of peppers are shown in r 4 days B. To zoo < 0.05) B. Aged b < 0.05) B. This m < 0.05) B, which  < 0.05) A.Paocai during fermentation [entation . Both glentation . Besidesentation .Lacticaseibacillus casei and Lactiplantibacillus plantarum ferment lactose through the phosphoenol pyruvate phosphotransferase system to produce glucose and galactose-6-phosphate [The glucose content in PAB and JAB increased and then decreased A, which hosphate . In addiFree amino acids are one of the main flavor substances in fermented vegetables, of which quality and quantity play important effects on the flavor quality . In this1 was the samples without fermentation, category B2 was the jar-fermented samples, and category B3 was the pool-fermented samples. It indicated that the effect of fermentation containers on the FAAs content of fermented chili peppers was more dominant than that of fermentation brines. Compared with the pickle jar, chili peppers fermented using the large-scale pool showed higher FAAs content, particularly for samples with aged brine (ed brine . In otheed brine . It mighLactiplantibacillus plantarum) through the secretion of peptide enzymes and the breakdown of protein into FAAs [The concentration of FAAs increased until day 4 and then decreased during fermentation . The chanto FAAs . The reanto FAAs . It coulnto FAAs . In addinto FAAs ,30.β-ocimene and limonene were the main terpenes detected in all fermented chili peppers, which contributed citrus and floral odors [Bacillus amyloliquefaciens, which imparts a sweet and pineapple aroma to fermented chili peppers [Debaryomyces hansenii [Representative total ion chromatograms of headspace volatile components of fermented chili peppers, using different brines and containers when fermented for 30 days are depicted in al odors . Compara peppers ,33. Styrhansenii .Y−variance. Since each LV explained an equal proportion of the Y−variance, all three plots are shown. Paocai fermented with aged brine was considered to have the most intense flavor and authentic taste [Considering the large dataset obtained, PLS−DA modeling was used to determine the correlation of fermentation methods with volatiles . During ic taste .In this study, the volatiles with an absolute VID value higher than 0.800 was selected as discriminant volatile compounds and represented by bold circles . Most diPichia fermentans and Pichia anomala, and Pichia fermentans and Pichia anomala were positively correlated with the content of 3-methylbutyl 2-methylbutanoate [As shown in utanoate .β-chamigrene, (Z)-β-ocimene, o-cymene, methyl-1-naphthalene, and durene, imparting a floral and herbal aroma to the fermented chili peppers [Metschnikowia pulcherrima is characterized by an extra-cellular α-arabinofuranosidase, which influences the content of terpenes in the fermented product [Metschnikowia pulcherrima seems to display a high aerobic respiratory metabolism and requires high levels of oxygen [Metschnikowia pulcherrima. The high content of most terpenes in the pool might be related to the high level of Metschnikowia pulcherrima.Terpenes were the second largest aroma compounds in fermented chili peppers . Most of peppers . Metschn product . Metschnf oxygen . In thisp < 0.05)  . SimilarPaojiao [p < 0.05)  . Our prethe pool . In addithe pool . The celthe pool . As aromIn the study, the effects of different brine (fresh/aged brine) and large-scale containers (pool/jar) on the taste and aroma formation of fermented chili peppers during natural fermentation were investigated. Considering the large datasets, clustered heatmap, PLS-DA modeling, and kinetic modeling were conducted to extract the key flavor compounds distinguishing chili peppers fermented using different procedures.p < 0.05). Sugar was consumed faster in chili peppers fermented by fresh brine than that by aged brine. Compared with the pickle jar, chili peppers fermented using the large-scale pool showed higher FAAs content, particularly for samples with aged brine, such as Asp, Thr, Ser, and Ala.As for taste properties, the results showed the effect of brine on organic acid and sugar profile was more dominant than that of containers, while FAAs content production was more affected by containers than brines. Specifically, chili peppers fermented using aged brine exhibited a significantly lower pH value and higher organic acid content than that fermented using fresh brine (p < 0.05), including 4-methylpentyl isobutyrate, heptyl hexanoate, amyl 2-methylbutyrate, isopentyl isovalerate, (Z)-3-hexenyl 3-methylbutanoate, heptadecane, hexadecane, octadecane, naphthalene, isohexanol, hexanol, acetic acid, 3,7-dimethyl-6-octenoic acid, and 3,5-dimethyl-2-octanone. Compared with the pickle jar, the content of terpenes in chili peppers fermented in the pool was higher, including β-chamigrene, (Z)-β-ocimene, o-cymene, methyl-1-naphthalene, and durene. In general, jar fermentation with aged brine contributed more flavor to pickled chili peppers than other procedures.As for aroma, more aromatic compounds were detected in chili peppers with aged brines. Among the samples, the PAB samples showed the highest numbers of volatile compounds (96), followed by the JAB (88), PFB (80), and JFB (76). These volatile compounds mainly include esters, terpenes, alkanes, alcohols, ketones, and acids. The esters, alkanes, alcohols, acids, and ketones of aged brine were significantly higher than those of fresh brine-fermented chili peppers (


Background and Objective: Obstructive sleep apnoea (OSA) is characterized by nocturnal repetitive upper airway (UA) collapse. For sleep physicians, the recognition of UA collapse characteristics is critical for understanding OSA mechanisms and developing individualized treatment plans. Drug-induced sleep endoscopy (DISE) is an exam during simulated sleep that allows the dynamic assessment of the UA of individuals with OSA. The initial recognition of DISE was to locate the sites of UA obstruction and direct the surgical selection of OSA since it was introduced in the 1990s. After approximately 30 years of studies, based on advances in endoscopic operative techniques and innovative treatments of OSA, DISE had been performed to explore mechanisms and comprehensive treatments related to UA collapse. Methods: This article reviewed contemporary DISE advances, including indications and contraindications, technique of induced sleep, endoscopic operation, UA characteristics classification.Results and Conclusions: Precise selection based on the association between collapse patterns and treatment modalities, such as continuous positive airway pressure, oral appliance, positional therapy, robotic surgery and neurostimulator implanting, is the future research prospect based on DISE.DISE provides sleep physicians with valuable information about the upper airway collapse characteristics and dynamic changes during sleep.The studies based on DISE findings improve the selectivity and efficiency of treatment modalities, including classical therapies such as continuous positive airway pressure, oral appliance, positional therapy, and innovative therapies such as neurostimulator implanting and robotic surgery, promote the advancement of OSA precision medicine In 1999The requirement for precisely evaluating UA collapse in individuals with OSA and improving surgical results promotes the appearance of drug-induced sleep endoscopy (DISE) in the 1990s. DISE is an exam that enables the dynamic study of the upper airway (UA) during sleep induced by drugs. Observation through DISE can find the anatomical deformity and muscular dysfunction with partial or complete collapse, involvement of one or more sites, and different configurations of UA collapse during episodes of apnoea and hypopnoea and can guide the choice of treatment based on various findings. After nearly two decades of research, DISE has made great progress in operative techniques and clinical guidance. The increasing use of propofol and target controlled infusion, with the Bispectral Index (BIS), capnography, oxygen saturation monitoring technologies, helped provide induced sleep similar to natural sleep. UA characteristics classifications were created and the association between the collapse patterns and the surgical success rate was found. Recently, DISE has also been performed on individuals without surgical indications, such as intolerance to continuous positive airway pressure (CPAP) therapy, suspected positional OSA and possible intraoral device candidates. A series of position papers has been published, moreover, more physicians have recognized the importance of identifying collapse characteristics through DISE .2., and fibre-optic sleep endoscopy [This procedure was first introduced as sleep nasoendoscopy. Several nomenclatures have been applied, such as video sleep nasoendoscopy, sleep endoscopyndoscopy . Howeverndoscopy , it prov3.DISE is a complementary exam in the evaluation of the patient with OSA and should never be used as a substitute for polysomnography (PSG) because it does not provide information about the types of respiratory events and the severity of OSA. The PSG should be accomplished before DISE.3.1.Indications include (1) patients with OSA and the need for non-CPAP treatment . (2) Pat3.2.Absolute contraindications include American Society of Anaesthesiologist (ASA) type IV: high-risk patients with severe systemic disorders that put the patient’s life at risk, Pregnancy, Previous history of allergy to sleep inducing-drugs. Relative contraindications include morbid obesity and tonsillar hypertrophy grade IV .4.2), electrocardiogram (ECG), and blood pressure (BP). A video-endoscopy system with a flexible nasoendoscope 4 mm in diameter or smaller can be used. Other suggested supplies and equipment include: (i) a standard infusion pump, preferably with target-controlled infusion (TCI). TCI is more effective and safer, allowing better adjustment of the infusion speed [Recommended monitoring during DISE includes oxygen saturation  with oxygen desaturation and respiration. It is recommended to record two complete cycles for each UA segment, including during manoeuvres, and the number of these cycles should be increased when the administration is performed in bolus .7.2 saturation during endoscopic observation. To avoid the effects of propofol overdose on neural pathway and muscle tone, it was recommended to use the TCI system and monitor the process of induced sleep to avoid severe hypoxaemia adverse effects [The isolated use of sleep-inducing drugs is the most common pattern . Fentany effects ,25. Corr effects ,27. MoreGiven the modes of medication administration  was usually required to maintain retrolingual airway patency, widely higher than at the retropalatal level (<10 cmH2O) [2O with each apnoea until the airway lumen achieves complete stabilization [DISE and CPAP can be performed simultaneously, which allows us to understand and identify unresolved UA collapse under CPAP therapy. It had been found that a higher CPAP pressure (>15 cmH0 cmH2O) . Moreove0 cmH2O) . The ass0 cmH2O) . The evalization . However10.The documentation of the examination record is recommended; usually, only videos or images of UA are stored. In 2017, Gobbi et al.  proposed11.DISE findings classification systems play important roles in clinical analysis, comparison, and treatment decision-making. It is recommended that a classification system should include at least the following characteristics: obstructive level (and/or structure), degree (severity of airway obstruction) and configuration (direction of collapse) . Thus faTwo classifications stood out earlier: VOTE  and NOHL  ,37. The In relation to obstructive structures in UA, VOTE uses sites , others use levels ,37,39. V12.Regarding to surgeries, based on the sites, configurations, and severity of UA collapse through DISE observation, it is possible to predict the outcome of UA surgery. Although the evidence supporting DISE prior to surgery was scarce and inconclusive , some stRegarding CPAP therapy, DISE was not recommended for titration, but could be used to identify the causes of CPAP failure. The flexible endoscope could be inserted into the mask through bronchoscopy adapter or from the underside of the mask, and then into the nasal cavity for DISE. While CPAP was turned on, UA dynamic changes under continuous positive pressure could be assessed. It had been found that the positive pressure required to maintain compliance of the lateral pharyngeal wall was lower than the anteroposterior collapse of the tongue base, then was velum or epiglottis . For patRegarding OA therapy (OAT), DISE showed to be a useful tool for screening responders, measuring the distance of mandible advancement, and improving OAT efficiency. In a clinical observative study, 35 patients with incomplete response to OAT  were performed DISE without and with OA in place, respectively. The proportion of velum, lateral pharyngeal wall, tongue base and epiglottis collapse decreased from 91%, 43%, 31%, and 31% at baseline to 43%, 11%, 9%, and 20% after wearing OA. The velum and epiglottis collapse had less improvement and more residual, thought to be associated with OAT failure . AnotherRegarding maxillomandibular advancement (MMA), DISE provided new insights into skeletal surgery for OSA. As mentioned above, baseline retropalatal CCC during DISE was considered a predictor of non-response or failure in soft tissue surgery or OAT. However, for MMA, CCC was not a contraindication. In a prospective case series study, DISE outcomes before and after MMA were compared. Pre-MMA retropalatal CCC were all completely alleviated, and the AHI improvement in individuals with CCC was equal to those without CCC . Based oRegarding trans-oral robotic surgery (TORS), DISE indicated that lateral velopharyngeal collapse was a potential Influencing factor for TORS tongue base reduction and/or multilevel surgery. Patients without lateral wall collapse had higher response rate (66.7%). Prospective studies with larger samples were expected to fully evaluate the predictive mode of DISE for TORS .Regarding hypoglossal nerve stimulation (HNS), DISE was considered a routine preoperative screening assessment. Moderate to severe OSA patients without CCC at the level of the soft palate were found to have satisfactory effects after long term follow-up. CCC had been widely accepted as a contraindication for neurostimulator implanting . However13.The pathophysiology of OSA is attributed to various factors, such as anatomy, central nervous system control, respiratory feedback and myopathy, ultimately manifesting as UA collapse during sleep. Although CPAP is the most common treatment, alternative therapies are also required for patients with low adherence and intolerance. Therefore, understanding the changes in UA compliance during sleep is very helpful for physicians to identify causes and then propose targeted treatment plans. DISE provides a unique viewing mode to directly examine continuous dynamic changes in the airway lumen during sleep. In the past 20 years, research on targeted therapy for DISE subtypes has increased year by year, and a series of position papers have been proposed. Through DISE, the phenotype of UA obstruction has been further refined according to various sites and collapse configurations, going beyond conventional definitions such as "pharyngeal cavity narrowing" or "tongue hypertrophy". Surgeons have become accustomed to using DISE to assess surgical indications and improve outcomes. At the same time, the complex regulatory mechanism of UA collapse has also been recognized, and future treatments are far from simply correcting anatomical deformities. The most interesting advances are combining DISE with conservative therapies  and novel therapies  to improve accuracy and effectiveness.DISE concurrent with wearing CPAP and OA can help physicians understand how UA obstruction is actually improving; whether the degree of improvement can reach expectation; and if it fails, what are reasons and what would be subsequent solutions. The manoeuvres of head rotation or protrusion of mandibular during DISE can predict the effectiveness of postural therapy or mandibular advancement therapy. Several innovative treatments have in turn increase acceptance of DISE among sleep medicine physicians, for example, the success of hypoglossal nerve stimulator implantation clearly demonstrating the necessity of DISE in screening candidates to exclude velopharyngeal complete concentric collapse. The complete lateral pharyngeal collapse can be restored after maxillomandibular advancement.However, there were some limitations of DISE. No studies have been able to identify the presence of REM sleep during DISE with midazolam, propofol and/or dexmedetomidine. Heo et al. proved that obstructive patterns may change if the observation duration is too long . HoweverCurrently, DISE is mainly performed in the operating room and requires sedation by an anaesthesiologist, post-anaesthesia monitoring by a nursing team, and endoscopic operation by an experienced otolaryngologist, which allows for more accurate and reliable judgement of obstructive sites, patterns, and degree . HoweverThere are clinical parameters such as sex, body mass index (BMI), race, drug addicts, type of bite and face, craniofacial deformities, neuromuscular and degenerative diseases, use of certain medications, severity of apnoea, percentage of desaturations and findings of endoscopic examinations with the patient in wakefulness that have been identified in the studies to date to correlate with DISE findings. For example, some findings in nasopharyngolaryngoscopy could predict DISE results, especially the fall of the epiglottis on forced inspiration. Authors showed that patients with CCC of the soft palate were significantly more likely to have higher AHI and BMI, while anteroposterior velar collapse was significantly associated with a lower BMI. Moreover, the AHI was significantly higher in patients with complete anterior-posterior collapse of the tongue. Lower AHI was associated with a higher probability of a partial concentric collapse ,55. ThesGenerally, compared to other dynamic examinations such as intraluminal pressure monitoring, cine CT or MRI, DISE has advantages in the balance of accuracy, operability, cost, and efficiency, it will remain the focus of OSA research. More prospective, randomized controlled studies are needed to guide the clinical practice. In the future, synchronous EMG and EEG monitoring during DISE will provide insight into the myopathy and neuropathy of airway collapse. The invention of tiny, less irritating instrument is expected to enable endoscopy in true natural sleep.14.Despite the limitations of DISE itself, it is undeniable that it has become the most common and most widely performed OSA evaluation method. DISE not only provides clinicians with valuable information about the sites, pattern and degree of obstruction but also provides profound insights into the UA collapse mechanism during sleep. Considering that DISE is closely involved with innovative therapies such as hypoglossal nerve stimulation and TORS, it is likely that DISE will play a more important role in guiding clinical practice in the future.AV and CZ contributed to the concept and design of the study. DE carried out the materials analyses. AV and DE contributed to the manuscript editing and writing. CZ contributed to the manuscript revising. All authors contributed to the manuscript approving.


Zea mays ssp. mays) is a major cereal crop worldwide and is traditionally or commercially cultivated almost all over the Americas. The North-Western Argentina (NWA) region constitutes one of the main diversity hotspots of the Southern Andes, with contrasting landscapes and a large number of landraces. Despite the extensive collections performed by the “Banco Activo de Germoplasma INTA Pergamino, Argentina” (BAP), most of them have not been characterized yet. Here we report the morphological and molecular evaluation of 30 accessions collected from NWA, along an altitudinal gradient between 1120 and 2950 meters above sea level (masl). Assessment of morphological variation in a common garden allowed the discrimination of two groups, which differed mainly in endosperm type and overall plant size. Although the groups retrieved by the molecular analyses were not consistent with morphological clusters, they showed a clear pattern of altitudinal structuring. Affinities among accessions were not in accordance with racial assignments. Overall, our results revealed that there are two maize gene pools co-existing in NWA, probably resulting from various waves of maize introduction in pre-Columbian times as well as from the adoption of modern varieties by local farmers. In conclusion, the NWA maize landraces preserved at the BAP possess high morphological and molecular variability. Our results highlight their potential as a source of diversity for increasing the genetic basis of breeding programs and provide useful information to guide future sampling and conservation efforts.Maize ( Genetic erosion is the main problem associated with the selection and improvement of agronomically important species. Despite the progress achieved in terms of productivity, modern breeding takes advantage of only a small fraction of the available variability, restricting the response of crops to pests, diseases and environmental changes –3. The uhttps://seedsofdiscovery.org/; www.amaizing.fr; The recent recognition of landraces as sources of diversity and beneficial alleles has been expressed through different initiatives aimed at providing an extensive characterization of germplasm bank collections. This characterization involves molecular and phenotypic aspects of maize and other crops [e.g., The high genetic diversity of maize landraces in the Americas has been thoroughly documented. e.g. –12. HoweThe northwestern region of Argentina (NWA) is the southernmost distribution limit of the Andean maize landraces. This region is characterized by a remarkable topographic variability, as it comprises six phytogeographic provinces located within a relatively limited area  . Two concirca 56 maize landraces described for northern Argentina are native to NWA, making it one of the main diversity hotspots of the Southern Andes [More than 50% of the rn Andes , 15. In rn Andes . Currentrn Andes . So far,rn Andes , 18. Thern Andes  on the brn Andes  and Vigorn Andes , 18.The present scenario of increasing food demand and climate change highlights the need for materials capable of growing under extreme conditions, such as the NWA landraces. Therefore, the characterization of the accessions in germplasm banks is important in tackling these challenges. Today, the “Banco Activo de Germoplasma de maíz in EEA-INTA Pergamino, Argentina” (BAP), preserves more than 2500 accessions from over 10 collections performed between 1977 and 1994. The almost complete absence of molecular information and the poor knowledge of the morphological variation and genetic merit of this vast collection  hinders in situ and ex situ conservation efforts.In this study, we present the morphological and molecular characterization of 30 accessions of BAP from NWA, which are representative of 17 NWA landraces and were collected at sites between 1120 and 2950 masl. They were sown in a common garden at 2300 masl and characterized for 19 morphological characters and 22 SSR loci to evaluate their agronomic potential, determine their genetic constitution and guide A set of 30 accessions from BAP were selected to encompass the morphological diversity occurring in NWA and cover a broad altitudinal range (collection sites between 1120 and 2950 masl) . All theAll the accessions were morphologically characterized at the Instituto de Pequeña Agricultura Familiar (IPAF), Hornillos, Jujuy Province, Argentina . One-hundred seeds per accession were sown under a randomized block design with two replicates. The elementary plots consisted of two rows of 5 m length, spaced 0.5 m apart. A conventional tillage was applied, with manual weeding and supplemental irrigation. No fertilizer or insecticide was added.https://www.bioversityinternational.org/e-library/publications/detail/descriptors-for-maizedescriptores-para-maizdescripteurs-pour-le-mais/), were scored. The following vegetative traits were assessed: plant height (PH), number of leaves (NL), number of leaves above the uppermost ear (NLA), uppermost leaf length (ULL), uppermost leaf width (ULW), venation index (VI) and tillering index (TI). The descriptors measured on the tassel were tassel length (TL), tassel peduncle length (TPL), tassel branching space (TBS), number of primary branches on tassel (NPBT), number of secondary branches on tassel (NSBT) and number of tertiary branches on tassel (NTBT). The variables recorded on the ear were uppermost ear height (EH), ear peduncle length (EPL), ear diameter (ED), mean number of ears per accession (MNE), number of rows of kernels per ear (NRK), and number of kernels per row (NKR).Nineteen agro-morphological descriptors, selected from the list of descriptors of the International Board for Plant Genetic Resources  and correlations between traits were computed using InfoStat 2018 .FactoMineR [factoextra [adegenet [Using the accessions as operational taxonomic units (OTUs), a Principal Component Analysis (PCA) was performed based on average trait values. To avoid biases related to the difference in scale between the variables, data were standardized so that their average was zero and the standard deviation was equal to one. R packages ctoMineR  and factctoextra  were usectoextra , employictoextra  to find adegenet .A total of 379 individuals were genotyped for the molecular analyses. The number of individuals per accession is given in ® 4.0 software  using a commercial size standard for allele size assignment . Automatic allele calls were subsequently confirmed reviewing all electropherograms.Twenty-two SSR loci were selected from a preliminary survey of 27, and only loci with unambiguous interpretation were used for this analysis . Genotypo) and expected (He) heterozygosities, allelic richness (Rs) [Mean number of alleles per locus (A), allele frequency, observed (Hess (Rs) , presencess (Rs) . Geneticess (Rs)  using Gehttp://www.pritch.bsd.uchicago.edu). The number of clusters evaluated ranged from 1 to 10. The analysis was performed using 10 replicates runs per K value, a burn-in period length of 500,000 and a run length of 1,000,000. No prior information on the origin of individuals was used to define the clusters and the correlated frequency model was used for all the analyses. The model assumes that the frequencies in the different populations are likely to be similar due to common ancestry [Genetic structure was examined using the Bayesian model-based approach of Pritchard et al.  implemenancestry . The delancestry  was usedancestry . Accessiancestry .mantel function of the R package vegan [ggplot2 [Correlations between genetic, morphological, and altitudinal distances were evaluated using Mantel tests . Analyses were conducted based on Spearman correlation coefficient using the ge vegan . We alsoge vegan . Signifi[ggplot2 .Global averages for the 19 quantitative agro-morphological traits scored in the study are presented in All the morphological characters analyzed showed considerable levels of variation across accessions . The coeThe analysis of association between variables revealed 56 significant correlations, which decreased to 20 after Bonferroni correction . Most ofAnalysis of morphological variation using the K-means algorithm allowed the identification of two main groups  . AccessiThe first group, G1 (black), presented a heterogeneous grain conformation, with accessions showing dent, semi-dent, sweet, flint and floury endosperm types . In thisOn the other hand, the G2 group comprised low-height accessions with pop, flint or semi-flint endosperm, fewer leaves, longer tassel peduncles, shorter branching space and very few tassel secondary branches. These accessions produced only a few ears per plant. The ears had smaller mean diameter and fewer grains per row than those in group G1, but a larger number of rows of kernels, resulting in a mean number of grains per ear of 350 .The third PC  maintained the same groupings, but further divided G1 into two sub-clusters, separating accessions with higher TPL and lower VI from the rest .o varied between 0.52 (CaG_1) and 0.65 (Pi_2), He between 0.63 (Pi_1) and 0.80 (B8H_2), and Rs between 3.77 (Pi_1) and 5.36 (B8H_2)  . A total (B8H_2) , with on (B8H_2) . All acc (B8H_2) .STRUCTURE-model-based approach allowed discrimination of different genetic clusters within the group of accessions examined here . Using tFor K = 2, most accessions could be assigned to one of the two groups, with an average proportion of membership higher than 0.85, except for A8H_1 and B8H_2, which showed an admixed composition , consistSpearman = 0.7, p <0.001), the pattern was not gradual, exhibiting an abrupt jump above 2000 masl. When these data were overlapped with the assignments found for K = 3, the accessions belonging to the yellow group corresponded to the lowest part of the altitude gradient, while those of the pink and blue clusters were cultivated above 2000 masl. Indeed, for K = 3, the mean altitude of the collection site of the accessions within the yellow, pink and blue clusters were 1684, 2584 and 2745 masl, respectively (A detailed examination of ectively .ST = 0.092 (p <0.01).The neighbor-joining network based on Nei’s distance was consistent with the partition into two groups inferred by STRUCTURE . In mostMorphological distances showed no correlation with genetic, geographic or altitudinal distances among accessions , as wellDespite the absence of correlation between morphological distance and altitude, the characters NL and NKR were negatively associated with the latter . LikewisEfficient use and conservation of the landraces available in germplasm banks requires comprehensive genetic and agronomic characterization focused primarily on variation at the level of individual accessions , 38–40. In this study, the clusters derived from morphological characters are consistent with those previously described for NWA landraces. Melchiorre et al. , 45 idenThe two major groups identified in the molecular analysis did not coincide with the morphological groups and showed a clear association with the altitude of collection sites, with the morphological group G2 being completely included in the highland group (>2000 masl). The levels of variability also appeared to go along with the differentiation of genetic groups, with a decrease in parameters such as expected heterozygosity and allelic richness in function of altitude. The lower variability of the highland group is consistent with the difficulties in growing maize under harsh environmental conditions , 43. In Regardless of how variability is distributed, NWA landraces taken as a whole show high levels of genetic variability, which are similar to the values recorded at the centers of crop diversity , 46, 47.Several studies have found a relationship between altitude of collection site and genetic composition of maize landraces both at the regional and continental levels , 10, 48.On the other hand, although morphological differentiation was not found to be correlated with altitude, individual trait analysis revealed a significant decrease for NL and NRK. Considering that all measurements were made in a common garden experiment at 2300 masl, these results may be interpreted as a local adaptation rather than phenotypic plasticity in response to environmental conditions. In fact, the relationship between flowering time, which is a key factor in the adaptation of landraces to altitude , 50, andOne of the main findings of our study is the remarkable differentiation between lowland (<2000 masl) and highland (>2000 masl) NWA accessions . In agreex situ conservation programs and to design future collection and management strategies. In particular, our results indicate that altitudinal structuring is a key factor in decision-making. For example, Perales et al. [in situ conservation strategies in lowland or intermediate elevation sites, where modern varieties have largely outcompeted native landraces. Despite the lack of similar studies in NWA landraces, it is clear that the altitudinal structuring of genetic diversity constitutes a relevant factor to be considered for their conservation.In conclusion, the present study constitutes a valuable contribution to existing s et al.  reportedS1 FigPC1 vs. PC3.(TIF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S6 Table(XLSX)Click here for additional data file.S7 Table(XLSX)Click here for additional data file.S8 Table(DOCX)Click here for additional data file.


H-1,2,3-triazol-1-yl)-9H-carbazole core. Similar to MBQ-167, MBQ-168 and EHop-097 inhibit activation of Rac and Rac1B splice variant and breast cancer cell viability, and induce apoptosis. MBQ-167 and MBQ-168 inhibit Rac and Cdc42 by interfering with guanine nucleotide binding, and MBQ-168 is a more effective inhibitor of P21-activated kinase (1–3) activation. EHop-097 acts via a different mechanism by inhibiting the interaction of the guanine nucleotide exchange factor Vav with Rac. MBQ-168 and EHop-097 inhibit metastatic breast cancer cell migration, and MBQ-168 promotes loss of cancer cell polarity to result in disorganization of the actin cytoskeleton and detachment from the substratum. In lung cancer cells, MBQ-168 is more effective than MBQ-167 or EHop-097 at reducing ruffle formation in response to EGF. Comparable with MBQ-167, MBQ-168 significantly inhibits HER2-positive tumor growth and metastasis to lung, liver, and spleen. Both MBQ-167 and MBQ-168 inhibit the cytochrome P450 (CYP) enzymes 3A4, 2C9, and 2C19. However, MBQ-168 is approximately 10× less potent than MBQ-167 at inhibiting CYP3A4, thus demonstrating its utility in relevant combination therapies. In conclusion, the MBQ-167 derivatives MBQ-168 and EHop-097 are additional promising antimetastatic cancer compounds with similar and distinct mechanisms.Rac and Cdc42, are homologous GTPases that regulate cell migration, invasion, and cell-cycle progression; thus, representing key targets for metastasis therapy. We previously reported on the efficacy of MBQ-167, which blocks both Rac1 and Cdc42 in breast cancer cells and mouse models of metastasis. To identify compounds with increased activity, a panel of MBQ-167 derivatives was synthesized, maintaining its 9-ethyl-3-(1Targeting the related GTPases Rac and Cdc42 that regulate cancer metastasis is a viable strategy to impede metastasis of solid cancers. Herein, we describe new Rac and Cdc42 inhibitors with unique mechanisms and varying potency in different cancer cell lines. The MBQ-167 derivatives MBQ-168 and EHop-097 show promise as potential antimetastatic cancer agents. Metastasis continues to be the leading cause of cancer-related deaths . Current50s of >10 μmol/L  of NSC23766 limits its use as a therapeutic agent. Our group designed EHop-016, a derivative of NSC23766, which inhibits Rac activity with an IC50 of 1.1 μmol/L by blocking the interaction of Rac with the GEF Vav  tumor growth and metastasis to all organs by approximately 90% in immunocompromised mice -9H-carbazole core was maintained with substitutions or isosteric replacements on the 5-phenyl group  were purchased from ATCC. GFP-tagged HER2-overexpressing metastatic cancer cell line (GFP-HER2-BM) characterized in ref. n = 3 biologic replicates: with three technical replicates each).Equal number of cells were seeded in a 24-well plate and incubated with vehicle or MBQ-167 derivatives at concentrations between 0 and 1,000 nmol/L for 72 and 120 hours. Using CellTiter 96 Non-Radioactive Cell Proliferation Assay  according to manufactures’ instructions, formazan was used as an indicator of viable cells at an absorbance 570 nm in a Tecan Microplate Reader M100 (Tecan). Data were calculated as fold change from vehicle and transformed to logarithm base 10. 0 concentration was replaced with 1 nmol/L to fit using the 4-parameter logic nonlinear regression model in GraphPad Prism software. .Equal number of cells were seeded in a 24-well plate and incubated with vehicle or MBQ-167 or derivatives at concentrations of 500 nmol/L for 48 hours. Apoptosis was measured using a Caspase-Glo3/7 Luminescence Assay Kit as per manufacturer's instructions , as described previously . Brieflyhttp://dev.mri.cnrs.fr/projects/imagej-macros/wiki/Wound_Healing_Tool; n = 3 biologic replicates: with three technical replicates each).MDA-MB-231 cells were plated on 12-well plates at equal cell density until confluent. Then the media was changed to 1% FBS, and a single scratch was made in the center of the monolayer culture with a pipet tip. Compounds were added immediately at 250 or 500 nmol/L and images were digitally acquired from a Keyence microscope (10×) at 0, 12, and 24 hours. The scratch area was quantified in ImageJ using the Macro Wound Healing Tool , were maintained under pathogen-free conditions in High Efficiency Particulate Air (HEPA)-filtered cages.5) cells in Matrigel (BD Biosciences) were injected at the fourth right mammary fat pad under isoflurane inhalation  to produce orthotopic primary tumors. After tumor establishment (1-week after inoculation), animals were randomly divided into treatment groups (n = 5).GFP-HER2-BM (∼7 × 10Mice were treated with vehicle  or 5 mg/kg Body Weight (BW) MBQ-167 or MBQ-168 by intraperitoneal injection in a 100 mL volume 3× a week. Treatments continued until sacrifice at day 53.2. To quntify metastasis, stored organs were thawed and analyzed, as whole organs on both sides, using an Olympus MV10 fluorescence macro zoom system microscope and images acquired with an Olympus DP71 digital camera. The fluorescent lesions (green component of RGB images) were quantified for integrated density of fluorescent pixels using Image J software.Mammary tumor growth was quantified by integrated density of GFP fluorescence. After tumor establishment, mice were imaged before starting treatment, and once a week thereafter for 53 days, using the iBoxScientia . Tumor fluorescence intensities were analyzed using ImageJ software . Relative tumor growth was calculated as the integrated density of fluorescence of each tumor on each day of imaging relative to the integrated density of fluorescence of the same tumor on day 1 of treatment. After sacrifice at day 60, lungs, kidneys, livers, and spleens were excised and immediately stored in liquid Nn = 3 biological replicates).Breast cancer cells were seeded at equal density and treated with vehicle, MBQ-167 or MBQ-167 derivatives for 24 hours at various concentrations. Rac-GTP and Cdc42-GTP were detected using Rac Activity Assay or Cdc42 Activity Assay, as per manufacturer's instructions . Briefly, total cell lysates were quantified and added equally to PAK-p21–binding domain conjugated Sepharose beads. After 1 hour of incubation, beads were washed, bound proteins extracted in sample buffer, and Rac1-GTP and Cdc42-GTP detected by Western blot analysis using Rac1, Rac1b, or Cdc42 antibody. , anti-Cdc42 (Abcam), anti-PAK1+PAK2+PAK3 , anti-pPAK1 T423)/PAK2 , anti-PAK1 + PAK2 + PAK3 , Rac1B, and β-actin (Millipore-Sigma). Actin was used as a control; the Rac/Cdc42 inhibitors do not affect total actin expression, only its polymerization and organization of the cytoskeleton . Drugs were diluted in exchange buffer and incubated with Rac1, Cdc42, or RhoA. The uptake of fluorescent nucleotide analog N-methylanthraniloyl-GTP (N-MAR-GTP) was measured as an increase in fluorescence due to binding to the nucleotide binding pocket of a GTPase. The fluorimeter was set at 20°C on kinetic mode and N-MAR GTP was detected at Ex 485 nm and Em 535 nm. After the initial five readings (T0), GEFs (Dbs or Vav2) or Ethylenediaminetetraacetic acid (EDTA) was added to stimulate exchange and readings were obtained every 30 seconds for at least 60 cycles.2, and protease inhibitors, and processed as described previously . The system contained recombinant human CYP enzyme and P450 reductase , luminogenic substrates appropriate for each CYP enzyme, a NADPH regeneration system (containing NADP+), and luciferin detection reagent. Control membranes devoid of CYP activity were used as negative controls. Ketoconazole, troglitazone and sulfaphenazole were used as positive controls. These compounds and organic solvents acetonitrile, and DMSO were purchased from Sigma-Aldrich.50 values.First, 12.5 μL of the 4X stock solution of test compounds or positive controls (appropriate for each enzyme) were added to the “treated” wells. For the “untreated”  and “minus-P450 control” wells , 12.5 μL of vehicle was added. Then, 12.5 μL of the 4X control reaction mixture  were added to the minus-P450 control wells, and 12.5 μL of the 4X reaction mixture  were added to all other wells. The plates were preincubated at room temperature for 10 minutes with the reaction mixture , and then 2X NADPH regeneration system was added to initiate the reaction. After incubation at room temperature for 30 minutes, 50 μL of the Luciferin Detection Reagent was added to stop the reaction and generate the luminescent signal. Before reading, the plate was incubated at room temperature for 20 minutes to stabilize the luminescent signal. The luminescence was measured using an Infinite F200 plate reader (Tecan). The values were displayed as relative light units. The percentage of CYP enzyme activity versus log concentration of test compounds were plotted to calculate the IC50) values were determined by using the 4-parameter logistic curve model in GraphPad Prism 9 software. Statistical analyses were done also using GraphPad Prism 9 software, and differences were considered statistically significant at P ≤ 0.05 using ordinary one-way ANOVA or unpaired Student t test with Dunnet multiple comparison tests.All half maximal growth inhibitory concentration  and MDA-MB-231 (327 nmol/L) cells, which were in the same range as the GI50 of 335 nmol/L for the MCF10A cell line. MBQ-167, MBQ-168, or EHop-097 did not affect the viability of HMECs indicating that these compounds are nontoxic to normal epithelial cells  data from a Contact Research Organization demonstrated no apparent toxicity from MBQ-167 in rodents or dogs up to 1,000 mg/kg. To determine whether the antiviability, antisurvival, and antimigration effects of MBQ-168 resulted in anticancer effects  vehicle  and B. N vehicle  and D. T vehicle . We did Because MBQ-168 is structurally and functionally similar to MBQ-167, we analyzed its mechanism of action as a Rac/Cdc42 inhibitor. Using pulldown assays via the p21-binding domain of PAK to isolate the GTP bound active Rac or Cdc42, we compared the efficacy of MBQ-167 to inhibit Rac and Cdc42 activation. First, the metastatic GFP-HER2-BM cell line was used to analyze the effect of 250 nmol/L MBQ-167 or MBQ-168 when attached and detached cells were collected following 24 hours incubation and subjected to pulldown assays for active Rac.GTP. Similar to MBQ-167, MBQ-168 affects cell polarity to result in cell detachment from the substrate to ultimately undergo apoptosis, that is, anoikis. We observed a complete depletion of Rac1-GTP bound MBQ-168 in both attached and detached cell population at 250 nmol/L in the metastatic GFP-HER2-BM cells. Notably, unlike with MBQ-167, Rac1 expression was approximately 100% decreased in the detached cell population in response to MBQ-168 treatment. As seen in the control Western for actin, which is also depleted, MBQ-168 appears to be a more potent apoptosis inducer and the detached cell population goes through anoikis at a faster rate than following MBQ-167 treatment. Furthermore, MBQ-168 was also more effective than MBQ-167 in reducing Rac1 activation (Rac1.GTP) in the attached cell population by approximately 100% in the GFP-HER2-BM cell line .50 of 500 nmol/L, which is 5× higher than the IC50 for Rac1 inhibition . The lysates were also probed for phosphorylation state of PAK 1/2/3 at the residues of threonine (Thr) 423 and serine (Ser) at S141 (PAK1), S144 (PAK2), and S154 PAK3; . For PAK MBQ-168 . TherefoThis inhibition in PAK expression followed by a more potent inhibition of phosphorylation in activation residues were also observed from tumor extracts. Tumors from the experiment shown in Like MBQ-167, 250 nmol/L EHop-097 significantly inhibited Rac1 activity by approximately 85% in MDA-MB-231 TNBC cells . When teTo identify the GEFs inhibited by MBQ-167 and derivatives, Sepharose-coupled purified Rac1(G15A) or Cdc42(G15A) a mutant form, which interacts tightly with GEFs was incubated with MDA-MB-231 cell lysates to recover GEFs bound to Rac1 or Cdc42, following preincubation with vehicle, or 250 nmol/L MBQ-167, MBQ-168, or EHop-097, as described in ref. To determine the mechanism of Rac1 and Cdc42 activation inhibition by MBQ-167 and MBQ-168, we monitored the loading of N-MAR-GTP to Rac1 in the presence or absence of MBQ-167 or MBQ-168. We recently reported that MBQ-167 acts as a GDP/GTP-binding inhibitor via nuclear magnetic resonance studies with purified Rac1. MBQ-167 addition resulted in a shift of the Glycine12 residue on Rac1, which is critical for interaction with guanine nucleotides . When thin vitro exchange assay , a family of hemeprotein isozymes primarily found in the liver . Therefoin vitro enzyme assay. The strongest inhibitory activity of MBQ-167 was seen on CYP3A4, followed by CYP2C19 > CYP2C9 > CYP1A2 (50 = 0.28 μmol/L), followed by MBQ-168 (IC50 = 3.18 μmol/L) > MBQ-169 (IC50 = 3.31 μmol/L) > MBQ-171 (IC50 = 8.04 μmol/L) > (IC50 = 15.03 μmol/L). MBQ-167 was also the most potent inhibitor of CYP2C19 with an IC50 of 0.455 μmol/L. This IC50 is even lower than that obtained with the positive control troglitazone (IC50 = 3.42 μmol/L). MBQ-168 and MBQ-169 also demonstrated IC50s lower than the positive control, with IC50s of 0.523 μmol/L and 0.58 μmol/L, respectively. Hence, these are strong CYP2C19 inhibitors. In terms of CYP2C9, the most potent inhibitor was MBQ-168 (IC50 = 0.67 μmol/L), followed by MBQ-169 (IC50 = 0.87 μmol/L) > MBQ-167 (IC50 = 0.89 μmol/L) > MBQ-171 (IC50 = 1.55 μmol/L) >  are direct downstream effectors of Rac and Cdc42 that have been the subject of intense investigation for targeted anticancer drugs due to their central role in cancer progression to metastasis . However50 for the transformed mammary epithelial cell line MCF10A was similar to the approximately 300 nmol/L GI50 in breast cancer cells. In contrast, MBQ-168 exhibited a higher GI50 in the MCF10A cell line, much as MBQ-167, achieving a low toxicity for this immortalized mammary epithelial cell line. In the ER/PR-positive MCF-7 cells, MBQ-168 demonstrated a lower GI50 compared with MBQ-167, therefore having a higher cytotoxicity effect in this cell line, which has more epithelial characteristics. Moreover, the primary human mammary epithelial cell line HMEC, was not inhibited by MBQ-167, MBQ-168, or EHop-097, thus demonstrating nontoxic effects of these compounds in nonimmortalized epithelial cells.The EHop-016 derivative EHop-097, a 1,4,5-trisubstituted-1,2,3-triazole derivatives was also selected as a potential compound for further analysis because this compound decreased MDA-MB-231 and GFP-HER2-BM cell viability and increased apoptosis. However, for EHop-097, the GIStudies on the effect of MBQ-167, MBQ-168, and EHop-097 on cancer cell migration and actin cytoskeletal extensions show that all three compounds reduce cell migration to a similar extent. The observation that MBQ-168 exhibit an antimigratory effect as MBQ-167, with decreased actin-based ruffles and migratory extensions in both breast and lung cancer cells combined with a loss in cell polarity and ultimate detachment from the substratum to result in cell death (anoikis), demonstrates that MBQ-168 is acting in a similar mechanism as MBQ-167. However, we found enhanced inhibitory performance for MBQ-168 in reducing actin cytoskeletal extensions in response to EGF in lung cancer cells. Therefore, MBQ-168 may be a more effective inhibitor of lung cancer progression than MBQ-167, which will be tested in future preclinical studies using lung cancer mouse models. In contrast, EHop-097 reduced actin cytoskeletal extensions without affecting cell polarity and was less effective at inhibition of EGF-induced actin ruffles in a lung adenocarcinoma cell line; demonstrating a different mechanism of action, as would be predicted by the structural and mechanistic differences of the compounds, as well as the different compliment of exchange factors that are active in lung cancer cells. For instance, it was recently reported that the A549 lung cancer cell line was dependent on the GEFs ARHGEF39, FARP-1, and TIAM-2 while the MDA-MB-231 and GFP-HER2-BM cells have a different GEF compliment and express Vav . TherefoSimilar to the effects on breast cancer cell viability and apoptosis, MBQ-168 inhibited tumor growth to the same extent as MBQ-167 in mice bearing GFP-HER2-BM mammary tumors. This drastic reduction in primary tumors was also reflected in reduced lung, liver, and kidney metastases to a similar extent by MBQ-167 and MBQ-168 demonstrating the feasibility of developing MBQ-168 as an additional anticancer compound. Future studies will determine the bioavailability and toxicity and/or safety of MBQ-168, as well as its effect in a range of cancer cell lines, for its continued translational development.Rac, Cdc42, and Rho activation assays conducted from cells preincubated with MBQ-167, MBQ-168, or EHop-097, demonstrated that these compounds were selective Rac/Cdc42 inhibitors without affecting Rho activation. MBQ-167 and MBQ-168 had similar inhibitory effects on Rac/Cdc42 activation. Total Rac1 levels were also reduced in tumors from mice that received MBQ-167 or MBQ-168 (5× a week for 2 months), indicating that prolonged treatment of MBQ compounds may affect Rac1 expression at the transcriptional or translational level. Protein stability is also a potential regulatory mechanism because Rac is also known to be ubiquitinated and degraded . We alsoBy monitoring the nucleotide loading of N-MAR GTP, we found that MBQ-168 was able to block GTP loading when catalyzed by the GEFs Vav2, Dbs or by intrinsic exchange using EDTA. Because we demonstrated that our compounds do not block the GEFs Vav2, Ect2, p-REX, Tiam1, etc., it can be construed that MBQ-167 and MBQ-168 impede nucleotide loading independent of the GEFs by binding the G12 residue and not a specific GEF-interacting site, as was shown previously for inhibition of the Vav/Rac interaction for EHop-016 , and in 2+/Mg2+ Chelator EDTA. The observed enhanced inhibition of EDTA-induced GTP loading on both Rac and Cdc42 by MBQ-168 compared with MBQ-167 demonstrates that MBQ-168 is more effective than MBQ-167. This superior efficacy of MBQ-168 may be attributed to MBQ-167’s poor solubility, pH sensitivity, or fluorescence interference due to the intrinsic fluorescence spectra of MBQ-167 rather than diminished efficacy because both MBQ-167 and MBQ-168 inhibit Rac activation from breast cancer cell lysates to a similar extent.As would be predicted by the action of an established guanine nucleotide association inhibitor , both MB50 that is 3-fold lower at the same concentration, while the other derivatives MBQ-168, -169, and -171 had higher IC50s. MBQ-168 was found to be approximately 10X less potent than MBQ-167 at inhibiting CYP3A4. This observation is relevant because CYP3A4 availability in tumors may limit the intracellular concentrations of docetaxel and paclitaxel, which are anticancer drugs that are substrates of CYP3A4  association with Tiam-1 and p-REX-1.Click here for additional data file.Suppl. Fig. S8Supplementary Figure S8 shows nucleotide loading of GTP binding to Rho GTPases.Click here for additional data file.


Simulation is an efficient approach for the investigation of theoretical and applied issues in population and quantitative genetics, and animal and plant breeding. In this study, we report a multi-module simulation platform called Blib, that is able to handle more complicated genetic effects and models than existing tools. Two derived data types are first defined in Blib, one to hold the required information on genetic models, and the other one to represent the genetics and breeding populations. A number of subroutines are then developed to perform specific tasks. Four case studies are present as examples to show the applications of Blib, i.e., genetic drift of multiple alleles in randomly mating populations, joint effects of neutral mutation and genetic drift, comparison of mass versus family selection, and choice of testers in hybrid breeding. Blib together with its application modules, has great potential to benefit theoretical genetic studies and intelligent breeding by simulating and predicting outcomes in a large number of scenarios, and identifying the best optimum selection and crossing schemes. Blib handles complex genetic effects and simulates large amounts of scenarios to identify the most optimum selection and cross schemes for intelligent breeding. On the other hand, simulation can generate a large amount of data that is impossible or difficult to obtain from the empirical experiments or theoretical considerations, which can be used to compare different selection methods, and validate the proposed theories or models. In addition, simulation can help perform pilot research, accelerate research and development, and transfer discoveries from the laboratory or theory to practice3. As an example, by using molecular data on parents and genomic prediction models, the segregating populations of virtual crosses can be generated and then compared, from which the most promising ones can be predicted before the crosses are actually made in the field4.Simplified assumptions are often made for reasons of tractability in many theoretical studies, however the real world is more complicated. Computer simulation provides an efficient tool that has been widely used in many scientific fields and disciplines, probably since computers were invented. Genetic studies and breeding applications allow us to investigate the implications of relaxing some of the assumptions made in population and quantitative genetics, and their effects on the activities of breeding programs6. Based on the QU-GENE platform, several application modules have been developed, and then used to investigate various issues in breeding. As an example module of QU-GENE, QuLine was designed to simulate the breeding programs for deriving inbred (or pure) lines and has been used to compare different breeding methods9, predict cross performance using known gene information10, optimize marker assisted selection in the pyramiding of multiple genes11, and investigate the efficiency of a single backcrossing breeding strategy12. QuHybrid is another module designed to simulate breeding programs for selecting hybrids as the final products, and it has been used to calculate the probability of success of a biofortified breeding program for improving the provitamin A content in maize13. More recently, QuMARS was developed to simulate and optimize various recurrent selection strategies, including phenotypic selection, marker-assisted recurrent selection, and genomic selection (GS) for both short-term and long-term breeding procedures14.To use the simulation approach in genetic studies and breeding applications, the development of suitable software packages, platforms and/or modules is fundamental. QU-GENE is such a platform that was developed for the quantitative analysis of genetic models15. PedigreeSim simulates meiosis in both diploid and tetraploid species and can be used to generate pedigrees and cross populations16. Slim is a forward simulation tool designed to study the effects of linkage and selection and is capable of modeling the complex scenarios of demography and population substructure17. Forqs is a forward-in-time simulator of recombination, quantitative traits and selection designed to investigate haplotype patterns in scenarios where substantial evolutionary change has taken place18. By using progeny simulation and genomic prediction strategies, PopVar can predict the population mean, genetic variance, mean of superior progenies, and correlated responses of multiple traits19. AlphaSim is a package for simulating plant and animal breeding programs that is flexible in terms of historical population structure and diversity, pedigree structure, trait architecture and selection strategy20. Similar to QU-GENE application modules, ADAM-plant can simulate breeding programs for self-pollinated and cross-pollinated crop plants, and for the application of new technologies such as speed breeding and GS21.Several other simulation platforms have been developed in past decades for various purposes. SimuPop provides a forward-time simulation environment, which may be used to manipulate genetic populations after many generations of random mating, and investigate the evolutionary mechanism of natural populationsThe QU-GENE platform together with its application modules represents one of the most mature and widely used simulation tools in genetics and breeding. The platform can handle a wide range of genetic models including molecular markers as well as genes underlying phenotypes, genetic linkage, multiple alleles, additive, dominance, epistasis, and gene-by-environment interactions. However, it was originally designed without considering more complicated effects and/or genetic phenomena, such as mutations, cytoplasm effects, and fertilities of female and male gametes, needless to say the interactions between cytoplasm and nuclear genes. These phenomena or effects are important in evolutionary biology, population genetics and breeding applications, but cannot easily be imbedded into the existing platform and its application modules.In this study, we report a multi-module genetic simulation platform called Blib, which has the potential for a much wider range of genetic studies and intelligent breeding. First, we introduce the technical issues for derived data types of the generalized genetic models and genetics/breeding populations, together with major subroutines in Blib. We then present four case studies as examples to show some of the applications of Blib in genetic studies and breeding.N) were set at 10 and 50. For each population size, four initial allele numbers were considered in the base populations, i.e., 2, 0.5 N, N and 2 N. The alleles had equal frequency in the base populations. The drift procedure was repeated for 100 generations of random mating and 100 simulation runs. For each generation, allelic frequencies in the 100 subpopulations (generated from the 100 simulation runs) were recorded.One Blib application module called DRIFT was developed to simulate genetic drift in randomly mating populations. First, two subroutines, i.e., Blib_ReadGmodel and Blib_ReadPopulation, were called to assign the global variable Gmodel and one initial population from two external files, respectively. Then, the subroutine Blib_Cross1Population was repeatedly called to conduct random mating in one parental population and generate the progeny population for a given number of generations. The whole procedure was repeated for a given number of runs or replications. Statistical parameters associated with the population were calculated by calling subroutine Blib_CalcPstatistics and outputted to external files for each generation of random mating and each run. One locus with multiple alleles but without mutation was considered in the genetic model. Two population sizes . Assuming there are a number of subpopulations under genetic drift starting from one base population, the observed inbreeding coefficient, i.e., FObs, can be estimated from the observed allelic frequencies in subpopulations, i.e.,pij is the observed frequency of the ith allele  in the jth subpopulation , and ith allele across the m subpopulations23.Equations  and 2)  2) are dns, Eqs.  and (2) k) occurring in the base population, even though k is included in Eq.  in the jth subpopulation .In practice, assuming there are a number of subpopulations under mutation and drift at generation N) were set at 10 and 50. For each population size, four initial allele numbers were considered in base populations, i.e., 2, 0.5 N, N and 2 N. All alleles had equal frequencies in the base populations. The mutation and drift procedure was repeated for 200 generations of random mating and 100 simulation runs. For each generation, allelic frequencies in the 100 subpopulations (generated from the 100 simulation runs) were recorded.In the simulation using the Blib application module DRIFT, one locus with multiple alleles and mutations was considered in the genetic model. Two rates at the level of pairwise mutation were considered, i.e., 0.0001 and 0.0002. Two population sizes  matrix, where k is the number of alleles at the locus ; S1 family selection, which is based on the performance of one generation of selfed families (S1); S2 family selection, which is based on the performance of two generations of selfed families (S2); and half-sib selection, which is based on the performance of half-sib families (HS). Forty cycles of recurrent selection were conducted. Upward selection was conducted for 20 cycles, followed by 20 cycles of downward selection.Two alleles were considered at each locus in the genetic model. Size of the initial population was set at 100. Models with three types of effects were simulated, i.e., pure additive effects (AD0), additive and dominant effects (AD1), and additive, dominant and epistatic effects (ADE). Ten independent genes were predefined to affect the trait of interest. For model ADE, five epistatic networks were considered. In each network, genes at two different loci interact with each other. Genotypic values at each individual locus or in each epistatic network are randomly assigned values between 0 and 1. Broad-sense heritability was set at 0.2 for each model. Mutation and no mutation were considered separately. When mutation was included, the rates of forward and reverse mutation were set at 0.02 and 0.01, respectively, for all genetic loci in consideration.23, where higher additive variance results in greater response to selection. Therefore, the population quickly moves to the peak trait value after a few cycles of selection under model AD0 with only the additive effects  are developed. Elite DH lines are selected through two stages of testcrossing evaluation. In stage one, the 10,000 DH lines are crossed with one tester from the other heterotic group, and 5% are selected on the basis of their performance in testing crosses. In stage two, the 500 DH lines retained from stage one are crossed again with the same tester, and 10% are selected, resulting in 50 lines which are then used to develop new hybrids and perform the next round of biparental crosses. The simulation was repeated for 100 runs.Assume one hybrid breeding program, such as that for maize, begins with two heterotic groups, each consisting of 50 inbred lines as parents. One target trait, such as yield, is controlled by 100 biallelic loci distributed on 10 chromosomes with known genetic effects, which may come from genotype-to-phenotype modeling of historical breeding data, training populations or any other genetic studiesDuring simulation, the 50 retained DH lines at the end of each breeding cycle are evaluated by the 50 crosses with the tester, 2500 single crosses with the 50 inbred lines in the other heterotic group, and the inbred lines per se. Figure 23. In this sense, hybrid breeding actually provides an example where the in-direct gain outperforms the direct gain.In hybrid breeding, the intensity of selection mostly comes from the selection on testing cross performance. However, the improvement of testing cross performance is in fact not the most important objective. Only a higher genotypic mean of single crosses with the alternative heterotic group can give breeders a greater opportunity to select more elite hybrids. Regarding the genotypic mean of 2500 single crosses, Tester1 is identified to be the best, and Tester4 is the worst Fig. . Genetic33.Predictions are expected at almost every stage during the rather long breeding procedure. When making crosses, breeders wish to know which parents should be chosen, and how many crosses should be made. In early segregating generations, breeders wish to know how many individuals should be planted in the field, how strong selection should be, and which phenotypic traits should be used as the selection criteria. At the stage of yield and adaptation testing, breeders wish to know the best locations for conducting the trials, how many locations should be used and how many replications should be arranged. Some issues of interest in breeding program design and analysis are beyond the reach of empirical experimental investigations. Simulation studies can be conducted to complement, extend, and improve the design and interpretation of empirical studies33. Once properly designed and developed, these tools can help investigate many what-if crossing and selection scenarios, allow many scenarios to be tested and compared in silico in a short period of time, and finally help breeders make important decisions before conducting resource-demanding field experiments11. Simulation and prediction tools are essential components especially for the incoming breeding 4.034, when breeding activities will be more driven by information technology in acquiring phenotypic data, molecular technology in acquiring genotypic data, and artificial intelligence in utilizing big data to make selection decisions.Generally, decisions can be made from predictions; predictions can be made from simulations. With the accumulation of our knowledge and understanding of the inheritance of breeding traits, it is anticipated that the simulation, prediction and decision-supported tools will be in high demand40, breeding modeling and simulation12, and computer tool and software development44. Blib is composed of one global variable to define the generalized genetic models, one derived data type to define the genetics/breeding populations, and a number of general subroutines for manipulation and calculation. Blib has been well-developed and widely tested in recent years, and it can be treated as a computing and programming library for the purpose of genetics and breeding simulation. By using the library as the underlying components, various application modules can be readily developed to complete specific tasks, which have been shown by the four case studies. The separation of application modules from the library can save a great deal of time and effort when other researchers wish to develop new simulation tools or modify the existing modules.We designed and developed Blib as reported in this study over our decades of research experience and advances in genetic analysis methodology6, Blib can define and handle mutations (both for cytoplasm and nuclear genes), cytoplasm effects, cytoplasm by nuclear gene interactions, and fertilities of female and male gametes. By considering the additional genetic effects and models not included in QU-GENE, we expect that the application modules built on Blib will have greater potential for the theoretical genetic studies and breeding practice. For example, theories on population genetics seldom consider more than two factors simultaneously due to the intractability in mathematical deduction. By using the Blib platform, an application module can be easily developed to evaluate the joint effect of multiple factors affecting population structure, such as drift, mutation, migration, selection, stratification, and admixture. Such a module can also be used to generate a large number of genetic populations to evaluate the efficiency of statistical methods in genetic linkage mapping44 and genome-wide association studies (GWASs), and evaluate the efficiency of various prediction models in GS33.In addition to genetic effects and models that have been considered in QU-GENEWe do not expect one or just a few application modules to be able to meet most purposes. In contrast, we expect that different application modules will need to be designed and developed for different purposes. However, one module can work with different models and simulate different genetic phenomena as defined in external input files. For demonstration and validation, we developed one Blib application module to simulate genetic drift in randomly mating populations , one module to simulate mass and family recurrent selection  and one module to simulate breeding programs for developing hybrids . As shown in Case studies I and II, by modifying the input information, the module DRIFT can simulate not only the randomly mating populations under genetic drift but also the populations under the joint action of mutation and drift. It can also simulate a single locus with two alleles, a single locus with multiple alleles, and multiple loci with two or multiple alleles. When multiple loci are considered in the genetic model, linkage can be defined as well. A similar situation is true for the PRS module shown in Case study III, when different kinds of genetic effects are defined in input files. By adopting this philosophy, we believe that the need to develop new modules can be minimized. For most researchers, their efforts can be spent more effectively in building suitable genetic models, designing simulation experiments, running suitable application modules, and analyzing simulation results.45. More application modules are being developed or planned, such as the in silico simulation of general breeding programs for developing pure lines, hybrids (as shown in Case study IV), and clonal varieties in plants, cross performance prediction and parental selection given the training populations with genotypic and phenotypic values, and simulation of various mapping populations from the user-defined linkage maps and genetic models. These modules are applicable to genetic studies and breeding practices in both sexually and asexually propagated species. Once developed and validated, all Blib-based application modules will become freely available to scientific researches.As another example, one specific module was developed to simulate the genetic recombination events between parents with the designed crossing schemes, which has been integrated with the genomic prediction tool to conduct genomic cross prediction in flax linseed breedingTo run one application module of Blib, at least three kinds of external files are needed as the input information. The first contains the information used to define the generalized genetic model , the second defines one or several initial populations, and the third defines the parameters in the simulation experiment. Although many genetic effects and models can be handled in Blib, not all of them have to be included in each external file or each simulation experiment. Take the application module used to simulate recurrent selections (i.e. Case study III) as an example. Three genetic models  are defined separately in three external files. To run the same module on the three genetic models sequentially, we can monitor the change in population mean due to phenotypic and family selection schemes, as shown in Case study III. By adopting this philosophy, we believe that the procedure used to develop the Blib application module can be greatly simplified and more straightforward. The development of application modules can only focus on the third kind of input information and organization of the Blib subroutines to complete more specific and complex tasks.46. By connecting with the information systems and ongoing breeding pipelines, the simulation platform Blib and its application modules can be used to conduct real-time simulation and prediction related to the outcomes of a large number of potential selection and crossing schemes. Thus, the optimum selection and crossing schemes can be identified and recommended to breeders before conducting field experiments. By doing so, simulation tools and approaches that are built on Blib are expected to make significant contributions by facilitating the change in conventional breeding from artificial selection to intelligent selection or helping move from the current stage to the incoming Breeding 4.0.To facilitate access to Blib and its application modules, we are currently developing some user interfaces that will help prepare the external files of input information for the global variable Gmodel and initial populations, which are fundamental to the simulation studies based on Blib. Alternatively, the information needed for simulation may be extracted from the existing database systems that are affiliated with on-going breeding programsIn Blib, a global variable named Gmodel was first defined, which holds all necessary information in a generalized genetic model. This information was packed up into 13 derived data types in Fortran ’ is first assigned with the size being equal to the number of genes defined in Gmodel%status. For each gene defined by ‘TYPE(Gmodel_gene)’ ’ is first defined, with the size being equal to the number of cytoplasms defined in Gmodel%status. For each cytoplasm defined by ‘TYPE(Gmodel_cyto)’ ’ is first defined with the size being equal to the number of cytoplasm actions defined in Gmodel%status. For each cytoplasm action defined by ‘TYPE(Gmodel_cact)’ ’ is first defined, with the size being equal to the number of fertility actions defined in Gmodel%status. For each fertility action defined by ‘TYPE(Gmodel_fert)’ (Supplementary Fig. In Blib, ‘Population’ is one derived data type in Fortran, which can be called to define the generalized genetic and breeding populations whenever needed (Supplementary Fig. The variable ‘…%pinput’ saves the frequencies of cytoplasm, and frequencies of alleles at each locus, or the specified allele combinations, which are loaded externally (Supplementary Table The other variables of derived data types record the genetic parameters at the population level, which are allocated and assigned upon request. The variable ‘…%stgv’ records population means, variances, genotype by environment interactions, broad-sense heritability, trait correlations, and environmental correlations. The variable ‘…%stvar’ records the additive and dominance variances, and broad-sense and narrow-sense heritabilities. The variable ‘…%stfre’ records the frequencies of cytoplasm, and frequencies of alleles at each marker or gene locus. The variable ‘…%stdiv’ records the diversities of markers and genes. Frequencies and diversities can also be calculated independently from genes located on each chromosome, from genes affecting each trait, or from genes in each epistasis network. All these parameters have been widely used in theoretical and applied population genetics.Based on the previously described global variable Gmodel and the derived data type ‘Population’, a number of general subroutines were then developed for specific purposes Fig. . Four suNine subroutines were designed to manipulate the population Fig. . The firOverloading is an important feature in modern programming languages. Five subroutines were designed to have the overloading features for populations (3.1–3.5 in Supplementary Table Three subroutines were developed for the purpose of generation advancement (4.1 to 4.3 in Supplementary Table Two subroutines were developed for the purpose of selection (5.1 to 5.2 in Supplementary Table All statistical analyses were performed using Microsoft Excel 2016. Case studies were conducted using the application modules and input files provided in Supplementary Software Further information on research design is available in the Supplementary Tables and FiguresDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Software 1Reporting Summary


There has been considerable debate about the extent to which our biological and linguistic histories match to each other, supported by examples of both matches and mismatches. We introduce a genomic database  to quantify matches and mismatches worldwide. While in most populations genetic and linguistic relations match, mismatches occur regularly as a result of language shift, and several language families follow diversification patterns different from that of the genomes. These findings reveal features of population contact in human history that were previously inaccessible to observation. Our database opens avenues for disentangling demographic and linguistic history and for comparing biological and linguistic modes of evolution. Human history is written in both our genes and our languages. The extent to which our biological and linguistic histories are congruent has been the subject of considerable debate, with clear examples of both matches and mismatches. To disentangle the patterns of demographic and cultural transmission, we need a global systematic assessment of matches and mismatches. Here, we assemble a genomic database  specifically curated to investigate genetic and linguistic diversity worldwide. We find that most populations in GeLaTo that speak languages of the same language family  are also genetically highly similar. However, we also identify nearly 20% mismatches in populations genetically close to linguistically unrelated groups. These mismatches, which occur within the time depth of known linguistic relatedness up to about 10,000 y, are scattered around the world, suggesting that they are a regular outcome in human history. Most mismatches result from populations shifting to the language of a neighboring population that is genetically different because of independent demographic histories. In line with the regularity of such shifts, we find that only half of the language families in GeLaTo are genetically more cohesive than expected under spatial autocorrelations. Moreover, the genetic and linguistic divergence times of population pairs match only rarely, with Indo-European standing out as the family with most matches in our sample. Together, our database and findings pave the way for systematically disentangling demographic and cultural history and for quantifying processes of shifts in language and social identities on a global scale. On the Origin of Species, Darwin went a step further and boldly proposed that the parallels were more than just conceptual. Famously, he claimed that “a perfect pedigree of mankind…would afford the best classification of the various languages now spoken throughout the world” , a large, high-resolution genomic resource designed for multidisciplinary research on human cultural and linguistic diversity. We use GeLaTo to address the following questions: How frequent are mismatches between genes and languages? Which scenarios can shape match and mismatch profiles? How genetically cohesive are language families? Within language families, do linguistic and genetic histories reflect the same temporal processes?Materials and Methods). Languages are, in turn, assigned to their language families based on a state-of-the-art classification . As genetic information, we use pairwise genetic distances estimated via the Weir and Cockerham STF , which includes ∼500,000 single nucleotide polymorphisms (SNPs) selected to be variable in populations from all the continents; the design of this chip makes it suitable for global scale genetic comparisons. Genetic populations are assigned to a geographic location based on where the samples were collected and to their main spoken language on the basis of linguistic and anthropological information (fication . Languagrham FST .SI Appendix). In response, we first assessed whether populations have enough neighbors from different language families so genetic and linguistic relations could be directly compared. Within a radius of 500 km, more than half of all populations in GeLaTo have a neighboring population in a different language family, and within a radius of 1,000 km, this proportion grows to ∼84%.We first quantified the extent to which genetic neighbors speak languages of the same family. This global-scale investigation is subject to constraints of genetic sample coverage, regional structure, and data availability across language families (Dataset S1). This suggests that mismatches are a regular outcome of language history and not just rare outliers.Across the whole dataset, we find that for most populations their closest genetic neighbor belongs to the same language family. However, a nonnegligible proportion (18%) is closest to a linguistically unrelated language (SI Appendix). Linguistic and genetic enclaves (or “matching enclaves”) have closer genetic and linguistic relatives in a distant region of the world . This strict heuristic can be calculated only for 20% (n = 79 of 397) of all populations in GeLaTo that are not directly related  to their neighbors. Moreover, the enclave scenario, as we propose it, does not take into account moderate amounts of gene flow that populations in contact might undergo.A total of 52 of these cases correspond to matching enclaves . 27 turn out to be genetic enclaves, and only one case is identified as a linguistic enclave. Examples of each of these cases are illustrated in STF distances within and between language families, restricted to the geographic span of the corresponding language family. If the transmission of genes and languages is mostly vertical, we expect the STF within families to be overall smaller than the STF between them and their neighbors. These populations are defined as genetically aligned with their linguistic relatives . Examples of aligned and misaligned populations are shown in STF within family larger than the median STF between families) is 20%, a proportion that is roughly robust after qualitative screening and consideration of potentially false positives  and after evaluating the effects of downsampling within families .In response to these limitations, we adopt a second heuristic that targets not individual enclaves but language families at a broader scale. We compare the distribution of SI Appendix, Figs. S7–S9). Hungarians are possibly one of the most studied cases of mismatch. They are genetically similar to their Indo-European speaking neighbors  but maintain a separate linguistic identity as a member of the Uralic family . In our dataset, they are the only case of a linguistic enclave . Their genetic profile can be described as a mix of ancestries from throughout the Mediterranean basin: genetically close to Eastern Sicilian, they share genetic relatedness also with Indo-European speakers from the Balkans and geographically distant Turkish and Middle East Afro-Asiatic speakers . Finally, previously described cases of language shift are less visible with the larger set of comparisons and higher genetic resolution that is available in GeLaTo. This is the case of the Armenians in the Caucasus. Mitochondrial genetic profiles from Armenian (Indo-European speakers) and Azerbaijan (Turkic speakers) have been described as closer to neighboring Caucasian populations  than to speakers of their respective language families , challenging the idea that they shifted their linguistic affiliation.Finally, we reviewed candidate misaligned populations against available genetic and historical literature, confirming cases of language shift and suggesting new mismatch cases . It remains unclear, however, whether these mismatches are exceptions to the norm within their language families , even when they represent misalignments or mismatches , or when sample size and geographic dispersion differ substantially between families . Dating methods in linguistics are controversial. For this reason, we use two kinds of linguistic time depths: one combining archaeological, historical, and linguistic quantitative comparisons , and one calculated using a recently introduced generalized Bayesian dating method (STF adjusted for effective population size (eN) and are calculated over those population pairs in GeLaTo that have a common ancestor at the root level of the linguistic phylogeny. We do not assume a systematic bias in overestimating or underestimating the genetic and linguistic times: for linguistic data, we operate within time depths that can be known or inferred. For genetic data, our eN calculations are approximate, but we filter for populations with stable population size, even if in the long-term different human groups experienced either expansions or decline.Linguistic time-depth estimates are reconstructed and calibrated on the basis of diverse types of archeological, historical, and linguistic evidence . Extant g method . GeneticNo family supports a particularly close match of divergence times . In someMaterials and Methods). Second, populations with divergent genetic histories can end up speaking linguistically related languages because of language shift (as in the case of genetic enclaves); in particular, admixture with divergent genetic ancestries can contribute to push the genetic reconstruction estimates back in time.These ancient divergent events suggest prehistorical mismatch events. Different explanations can contribute to this pattern. First, the discrepancy could be due to the lack of power in reconstructing deep genetic and linguistic estimates, which might be associated with larger uncertainty, especially for the genetic time split . SI Appendix, Fig. S23). Specific clade correspondences are found in all families, also in the Austronesian and Turkic trees, which are genetically noncohesive . Vertical parallel inheritance is represented by a 1:1 correspondence . By contrast, the Austronesian tree is characterized by an overall older divergence time for the genetic estimates, except for the Polynesian branch, where genetic and linguistic estimates are closer  than mismatches, but single-population mismatches are present in each continent and within each language family. Mismatches are, therefore, not exceptional events but rather regular outcomes of human history. The most common pattern among mismatches is the genetic enclave, where a population shifts to the language of a geographically neighboring but genetically distant population .At the level of language families, we find that about half of those for which we have sufficient data are genetically cohesive. Genetic cohesiveness, defined as a tendency for closer genetic distances between populations of the same language family, is in line with demographically induced spreads as proposed by the farming/language dispersal hypothesis , 9. HoweMaterials and Methods, mismatches can be also be assessed at shorter time scales , a panel of genetic diversity with linguistic identifiers. The genetic data analyzed consists of 597,573 SNPs typed with the Human Origins Array, an SNP chip designed to minimize the effect of ascertainment bias in worldwide human diversity . We included only populations with a minimum of five individuals for a total of 397 populations and 4,030 individuals, and we excluded sex chromosomes to not bias the analysis with the female to male ratio . All the genetic populations considered are matched with a unique Glottocode identifier . Analysis based on the STF distributions is calculated with the exclusion of 18 “drifted” populations that have both continental median STF and global median STF above 0.1 and therefore may have experienced genetic drift due to reduced population size and/or isolation. Dataset S1 collects relevant information associated with each GeLaTo population considered, together with their genetic characterization and diversity values: the median STF with all the other populations of the dataset, the median STF within macro regions, the number of neighbors from a different language family within the 1,000-km radius, the lowest percentile of the STF distribution associated with a mismatch, the closest STF between and within language family, the associated geographic distance, and the median of the STF between and within language family. The difference in the medians between and within language families is also annotated with relative CI. Each population is finally flagged as an enclave, a matching enclave, or as misaligned in their STF distribution. Dataset S2 collects pairwise distance measures . We compared STF distances between all pairs of a given language family and between language families. Linearized STF [equivalent to STF/(1 − STF)] is used for this analysis, following the IBD correlational hypothesis  regressions, neighbor-joining trees, quartet analysis, and data visualization are processed in R (see pothesis , as it rSI Appendix, SI Text. Additionally, divergence times with associated credible intervals for the root of major language families are reconstructed from generalized Bayesian dating (Time depth of language family is calculated from historical linguistics sources, associated with broad indicative time ranges. A list of references considered is available in n dating .Our second set of analyses consists of within-language family comparisons and includes linguistic distances, as linguistic divergence times extracted from summary trees generated through Bayesian analysis from previous publications , 48, 49.SI Appendix, SI Text.Further information on the methods employed is available in Supplementary FileSupplementary FileSupplementary File