Molecular Differentiation Research Articles

Molecular Differentiation of Cathepsins B and D, and of p53 Protein, and their Quantitative Assay in Biological Samples

Cathepsins B, D and protein p53 are three of the biomarkers with a large spectrum of implications in the field of physiopathology as many diseases proved to be associated with their variation in concentration. Therefore, there is a need for reliable tools able to perform molecular recognition/differentiation, and also reliable quantification of cathepsins B, cathepsin D, and protein p53. Two stochastic microsensors based on diamond/graphene nanopowders modified with 2,3,7,8,12,13,17,18-octaethyl-21H,23H-porphine manganese (III) chloride were proposed for the simultaneous differentiation and quantification of cathepsins B, D and protein p53 in whole blood, urine, saliva, and tumoral tissues. The lowest limits of determination and the highest sensitivities were recorded when the stochastic microsensor based on nanopowder of graphene was used. Wide linear concentration ranges were recorded for both microsensors. The recovery values obtained for the microsensors, were higher than 97.00%, and the relative standard deviations values were lower than 1.00%, when used for screening of whole blood, urine, saliva, and tumoral tissues.

Towards elucidating species diversity of European inlandStrigamia(Chilopoda: Geophilomorpha): a first reassessment integrating multiple lines of evidence

AbstractStrigamia centipedes are widespread in European forest soils. However, a complex and inconsistent taxonomy has developed over time. Based on a modern species concept, we evaluated multiple lines of evidence for speciation among inland populations of Strigamia from the Italian region to the Baltic region, across central Europe. Hypotheses of species delimitation were drawn independently from: (1) morphological differentiation, by means of model-based cluster analyses of 52 specimens, after controlling for allometry and sexual dimorphism; (2) syntopy of morphologically distinct individuals, assumed to be representatives of coexistent species, from > 700 sites; and (3) molecular differentiation, by various methods applied to cytochrome c subunit I sequences of the same 52 specimens. Diagnoses and geographical distributions were revised by re-examining > 2000 specimens and the entire literature. We found preliminary evidence for: a Strigamia acuminata species complex, widespread, including a candidate species from the Eastern Alps differing in the forcipules; a Strigamia carniolensis species complex, hitherto often called Strigamia crassipes, widespread south of the Alps but absent from Sicily; a Strigamia crassipes species complex, hitherto often called Strigamia transsilvanica, more widespread than previously known, including three candidate species co-occurring in the Western Alps and differing in body size and the number of legs despite little genetic divergence; and Strigamia engadina, exclusive to a narrow part of the Central Alps.

One genus, four different stories: evolutionary history, bionomics and biogeography of the monotypic tribe Stenostomatini (Coleoptera: Oedemeridae)

Abstract The false blister beetle tribe Stenostomatini includes only the genus Stenostoma, with four species showing an intriguing distribution: Stenostoma lowei (Madeira), Stenostoma cossyrense (Pantelleria), Stenostoma melitense (Malta and southern Sicily) and Stenostoma rostratum, widely distributed along the Mediterranean and North Atlantic coasts. The evolutionary history leading to this distribution has not been investigated. Here, we explore the phylogeny and evolutionary history of the tribe, adopting an integrative approach that combines morphological and molecular data (mitochondrial COI and nuclear CAD and 28S). Moreover, we propose a new key for species identification and update the knowledge on adult and larval ecology. Finally, we propose the following explanation for the current distribution of the species. The genus originated in the Miocene, with S. lowei belonging to the oldest lineage. The remaining species share a common ancestor, dating to the Messinian Salinity Crisis. Stenostoma melitense and S. cossyrense are well differentiated according to morphology, but not according to molecular analysis. The wide distribution of S. rostratum might be related to its strategy of laying eggs within beached driftwood, where larvae develop, easily transported by the currents. Given that neither morphological nor molecular differentiation was detected between the two subspecies of S. rostratum, we propose the new synonymy Stenostoma rostratum septentrionaleŠvihla, 2005 = Stenostoma rostratum (Fabricius, 1787) syn. nov.

Abstract P3040: Development Of A Protein Synthesis Network At The Sarcoplasmic Reticulum In Adult Cardiomyocytes

Introduction: The endoplasmic reticulum (ER) is the site of synthesis and folding of membrane and secretory proteins. In striated muscle cells, a specialized membrane system known as the sarcoplasmic reticulum (SR) sequesters Ca 2+ and carries key functions in excitation-contraction coupling. Textbooks often define the SR as the ER of muscle, but surprisingly, the biochemical and physiological relationship between the ER and SR and even whether they are distinct membrane networks or refer to the same structure remain unknown. Methods: To characterize the relationship between the ER and SR, we applied biochemical and imaging techniques, including immunofluorescence and election microscopy, fluorescence in situ hybridization, and STimulated Emission Depletion (STED) microscopy to ventricular myocytes, which allowed us to follow the differentiation of these membrane compartments in developing cardiomyocytes. Results: Using localization and expression levels of SERCA2, RyR2, KDEL and different markers of ribosomes, mRNA, and the secretory pathway, we found that the membrane compartment that exists in the early postnatal stage, resembles mostly the ER, and this membrane system decreases in postnatal development. The SR is the main membrane network that exists in the adult cardiomyocytes, replacing the ER, in all but a small perinuclear region. Both sites which appear to be interconnected, perform specialized tasks, with protein secretion through the classical ER-Golgi secretory pathway originating mainly from the perinuclear ER and Ca 2+ -dependent excitation-contraction coupling originating exclusively from the SR. Other functions, such as Ca 2+ intake or posttranslational protein processing occur in both compartments. Importantly, we show that in adult cardiomyocytes ribosomes are not only attached to the ER but also to the SR, where they actively perform mRNA translation and are major sites of protein synthesis. Conclusion: The ER and SR are two distinct coexisting membrane networks in cardiomyocytes that undergo molecular differentiation and structural organization during cardiac muscle development. Significant amounts of protein synthesis occur in adult cardiomyocytes at the SR and may serve localized mRNA translation near the sarcomere.

Molecular and morphometric differentiation of secondary filariasis vector Coquillettidia mosquitoes (Diptera: Culicidae) in Thailand.

Coquillettidia mosquitoes are important nuisance-biting pests and a vector of brugian filariasis in Thailand. However, comprehensive information about these mosquitoes remains unavailable such as molecular and morphometric differences among species. The lack of vector knowledge on Coquillettidia species could affect future disease control. This study aims to investigate differences in molecular variations based on mitochondrial cytochrome oxidase subunit I (COI) gene and wing geometric traits of three Coquillettidia species, namely Cq. crassipes, Cq. nigrosignata, and Cq. ochracea in Thailand. The results of molecular analyses revealed the differences among three Coquillettidia species. The genetic difference measure based on the Kimura two-parameter model among three Coquillettidia species showed low intraspecific distances (0%-3.05%) and large interspecific distances (10.10%-12.41%). The values of intra- and inter-genetic differences of three Coquillettidia species did not overlap which showed the existence of a barcoding gap indicating the efficiency of the identification based on the COI gene. As with molecular analysis, the landmark-based geometric morphometrics approach based on wing shape analysis indicated three distinct species groups which were supported by the high total performance score of cross-validated classification (97.16%). These results provide the first evidence of taxonomic signal based on molecular and wing geometric differences to support species identification and biological variations of Coquillettidia mosquitoes in Thailand for understanding these rare vector mosquitoes in depth and leading to effective further mosquito control.

Acute bee paralysis virus field isolates from apiaries suffering colony losses in Türkiye

Multiple viral infections are one of the main drivers of honey bee colony losses and threaten the sustainability of colony health. The Acute bee paralysis virus (ABPV) is one of the common honey bee viruses that can induce overt and/or covert infections in bee colonies. Globally more knowledge is needed about its effects leading to colony losses. ABPV-related pathology includes paralytic, trembling bees, unable to fly and, in some cases, ABPV causes the death of the bee colonies. The molecular phylogenetic of ABPV isolates obtained from apiaries suffering colony losses and/or with CCD-like complaints in Türkiye was performed in this study. The isolates’ structural and non-structural gene regions were analyzed phylogenetically and compared according to sampling years and apiaries. According to the results, the nucleotide identity of the RNA-dependent RNA polymerase (RdRp) gene in Turkish ABPV field isolates is more conserved than the capsid gene. The evaluation of molecular differentiation among the ABPV field isolates can be used for determining the origin and relatedness of ABPV strains and periodic monitoring of ABPV infections in apiaries.

Phenotypic and molecular differentiation of Lactococcus garvieae and Lactococcus petauri isolated from trout

Lactococcosis caused by Lactococcus garvieae leads to infective endocarditis, urinary tract infections, and peritonitis for humans and massive deaths for rainbow trout, an important zoonotic disease affecting fish, other animals and humans. However, the description of Lactococcus petauri revealed that most L. garvieae isolates belonged to L. petauri by genome analysis. In the present study, we revealed a comprehensive genomic comparison between L. garvieae and L. petauri species covering the determination of genome characteristics, overall genome-relatedness indices, genomic islands including virulence genes and antimicrobial resistance models, protein families, habitat preference and ecological distribution features. Besides, L. garvieae and L. petauri strains were also evaluated by whole-cell MALDI-TOF MS analysis. Notably, we assessed a species-specific primer design, named LG_IBS and LP_IBS, based on the genome analysis to distinguish L. garvieae and L. petauri for the first time by basic PCR. In addition to genotypic analyses, 177 L. garvieae isolates and four reference strains were tested to ferment sucrose which was determined as the main phenotypic test to differentiate these species for the first time. Our study revealed that L. garvieae and L. petauri have unique genes, and the designed primers, LG_IBS and LP_IBS, clearly differentiate both isolates. In phenotypical comparison, L. garvieae was observed as negative for sucrose fermentation, whereas L. petauri gave a positive reaction. Therefore, we confirmed that the sucrose fermentation test and designed PCR primers have 100% differentiation power for the isolates from two different laboratories for the first time. Moreover, a comparative analysis of protein families for L. garvieae and L. petauri strains revealed that the genomes of L. garvieae strains do not encode the genes required for sucrose metabolism, whereas L. petauri genomes encode genes for fructokinase, sucrose operon repressor and sucrose-6-phosphate hydrolase. Although both species exhibit similar habitat preferences, they also differ in the distribution of various genomic islands, including virulence factors and antimicrobial resistance genes. In conclusion, we propose that sucrose fermentation and newly designed primers yielding different amplicons for L. garvieae and L. petauri could be employed to differentiate the members of both species.

Integrative revision of the Blommersia wittei complex, with description of a new species of frog from western and north-western Madagascar.

Frogs of the Blommersia wittei complex are widespread in western and northern Madagascar, and are one of two clades of the family Mantellidae that have colonized the Comoran island of Mayotte. Based on a comprehensive set of DNA sequences of the mitochondrial 16S rRNA gene and the nuclear-encoded RAG1 and SACS genes, integrated with morphological and bioacoustic data, we here analyze the genetic differentiation of populations of this complex across Madagascar. We confirm that a candidate species named B. sp. Ca5 in previous studies represents a genetically well-defined evolutionary lineage distributed over much of western Madagascar, which we describe herein as Blommeria bara sp. nov. based on its molecular and bioacoustic differentiation. Blommersia wittei occurs across northern Madagascar but its type locality Ambanja, at the lower Sambirano river, is very close to the range of another, newly discovered microendemic lineage that was only found at two sites along the upper Sambirano river (here named as candidate species B. sp. Ca12). The B. wittei complex thus provides an example of a clade of closely related Malagasy frogs that contains species widespread over hundreds of kilometers, as well as extreme microendemics. For a full resolution of this species complex, more data need to be collected on the geographical contact among these two lineages, on the morphology and bioacoustics of B. sp. Ca12, and on the north-eastern populations of B. wittei at Sambava, which are weakly differentiated in mitochondrial genes but differ in bioacoustics and possibly in the extent of foot webbing.

Adaptative response to changes in pyruvate metabolism on the epigenetic landscapes and transcriptomics of bovine embryos

The epigenetic reprogramming that occurs during the earliest stages of embryonic development has been described as crucial for the initial events of cell specification and differentiation. Recently, the metabolic status of the embryo has gained attention as one of the main factors coordinating epigenetic events. In this work, we investigate the link between pyruvate metabolism and epigenetic regulation by culturing bovine embryos from day 5 in the presence of dichloroacetate (DCA), a pyruvate analog that increases the pyruvate to acetyl-CoA conversion, and iodoacetate (IA), which inhibits the glyceraldehyde-3-phosphate dehydrogenase (GAPDH), leading to glycolysis inhibition. After 8 h of incubation, both DCA and IA-derived embryos presented higher mitochondrial membrane potential. Nevertheless, in both cases, lower levels of acetyl-CoA, ATP-citrate lyase and mitochondrial membrane potential were found in blastocysts, suggesting an adaptative metabolic response, especially in the DCA group. The metabolic alteration found in blastocysts led to changes in the global pattern of H3K9 and H3K27 acetylation and H3K27 trimethylation. Transcriptome analysis revealed that such alterations resulted in molecular differences mainly associated to metabolic processes, establishment of epigenetic marks, control of gene expression and cell cycle. The latter was further confirmed by the alteration of total cell number and cell differentiation in both groups when compared to the control. These results corroborate previous evidence of the relationship between the energy metabolism and the epigenetic reprogramming in preimplantation bovine embryos, reinforcing that the culture system is decisive for precise epigenetic reprogramming, with consequences for the molecular control and differentiation of cells.

Combination of Internal Extractive Electrospray Ionization Mass Spectrometry and Statistical Analysis for High-Throughput Molecular Differentiation of Rehmannia glutinosa Samples.

Rehmannia glutinosa (Gaert.) Libosch. ex Fisch. et Mey. is a perennial herb of the Scrophulariaceae family, which has long enjoyed a good reputation in China, and has a wide range of pharmacological effects and clinical applications. The place of origin is an important factor affecting the chemical composition of R. glutinosa, resulting in different pharmacological effects. Herein, internal extractive electrospray ionization mass spectrometry (iEESI-MS) combined with statistical techniques was established for high-throughput molecular differentiation of different R. glutinosa samples. Dried and processed R. glutinosa samples from four different places of origin were analyzed by iEESI-MS with high throughput (>200 peaks) and rapidness (<2 min/sample) without sample pretreatment. Clear separation models created by OPLS-DA were then established for distinguishing the places of origin of dried and processed R. glutinosa by using the obtained MS data. In addition, the molecular differences between the pharmacological effects of dried and processed R. glutinosa were also investigated by OPLS-DA, and 31 different components were screened out. This work provides a promising method for evaluating the quality of traditional Chinese medicines and studying the biochemical mechanism of processing.

Non-cell-autonomous regulation of interneuron specification mediated by extracellular vesicles.

Disruption in neurogenesis and neuronal migration can influence the assembly of cortical circuits, affecting the excitatory-inhibitory balance and resulting in neurodevelopmental and neuropsychiatric disorders. Using ventral cerebral organoids and dorsoventral cerebral assembloids with mutations in the extracellular matrix gene LGALS3BP, we show that extracellular vesicles released into the extracellular environment regulate the molecular differentiation of neurons, resulting in alterations in migratory dynamics. To investigate how extracellular vesicles affect neuronal specification and migration dynamics, we collected extracellular vesicles from ventral cerebral organoids carrying a mutation in LGALS3BP, previously identified in individuals with cortical malformations and neuropsychiatric disorders. These results revealed differences in protein composition and changes in dorsoventral patterning. Proteins associated with cell fate decision, neuronal migration, and extracellular matrix composition were altered in mutant extracellular vesicles. Moreover, we show that treatment with extracellular vesicles changes the transcriptomic profile in neural progenitor cells. Our results indicate that neuronal molecular differentiation can be influenced by extracellular vesicles.

Costimulatory domains direct distinct fates of CAR-driven T-cell dysfunction

AbstractT cells engineered to express chimeric antigen receptors (CARs) targeting CD19 have demonstrated impressive activity against relapsed or refractory B-cell cancers yet fail to induce durable remissions for nearly half of all patients treated. Enhancing the efficacy of this therapy requires detailed understanding of the molecular circuitry that restrains CAR-driven antitumor T-cell function. We developed and validated an in vitro model that drives T-cell dysfunction through chronic CAR activation and interrogated how CAR costimulatory domains, central components of CAR structure and function, contribute to T-cell failure. We found that chronic activation of CD28-based CARs results in activation of classical T-cell exhaustion programs and development of dysfunctional cells that bear the hallmarks of exhaustion. In contrast, 41BB-based CARs activate a divergent molecular program and direct differentiation of T cells into a novel cell state. Interrogation using CAR T cells from a patient with progressive lymphoma confirmed the activation of this novel program in a failing clinical product. Furthermore, we demonstrate that 41BB-dependent activation of the transcription factor FOXO3 is directly responsible for impairing CAR T-cell function. These findings identify that costimulatory domains are critical regulators of CAR-driven T-cell failure and that targeted interventions are required to overcome costimulation-dependent dysfunctional programs.

Typing of Uncultured Isolates of &lt;i&gt;Coxiella burnetii&lt;/i&gt; and &lt;i&gt;Coxiella&lt;/i&gt;-Like Microorganisms Associated with Ticks Using &lt;i&gt;16S&lt;/i&gt; rRNA Gene Nucleotide Sequence Analysis

The causative agent of Q fever, the intracellular pathogen Coxiella burnetii, is found almost worldwide; many types of blood-sucking ticks that are dangerous to animals and humans are involved in the circulation of the pathogen. Using molecular-genetic methods, closely related species of microorganisms of the genus Coxiella sp. have been discovered, some of which are endo-symbionts of ticks, and some can survive in the human body, causing an infectious process. The existence of species whose genes are similar in nucleotide sequence to those of C. burnetii makes it difficult to diagnose the pathogen in arthropod vectors. The aim of this work was to consider the use of PCR and sequencing of an extended 16S rRNA gene fragment for molecular diagnostics and differentiation of C. burnetii from Coxiella-like microorganisms. Materials and methods. Individual samples of blood-sucking ticks were examined to detect bacteria of the genus Coxiella sp. applying standard PCR. For positive samples, an extended fragment of the 16S rRNA gene was obtained and examined by sequencing and multiple alignment with homologous sequences. Results and discussion. Of the 96 examined ticks collected in the Ulyanovsk Region, one was positive for the presence of C. burnetii DNA and one – for the presence of Coxiella sp. The greatest similarity for the C. burnetii isolate was noted in comparison with Western European strains, for the Coxiella-like microorganism - with closely related bacteria from ticks of the same species. Unique polymorphisms for the detected microorganisms were identified. It has been established that genus-specific primers to the 16S rRNA gene fragment are able to amplify not only bacteria of the genus Coxiella sp., but also genetically distant species. Analysis of the sequence of the extended 16S rRNA gene fragment makes it possible to differentiate C. burnetii from Coxiella-like microorganisms; some gene polymorphisms appear to have arisen through microevolution in different geographic regions. In the European part of the Russian Federation, Coxiella-like bacteria have been uncovered for the first time.

Splitting one species into 22: an unusual tripling of molecular, morphological, and geographical differentiation in the fern family Didymochlaenaceae (Polypodiales).

The pantropical fern genus Didymochlaena (Didymochlaenaceae) has long been considered to contain one species only. Recent studies have resolved this genus/family as either sister to the rest of eupolypods I or as the second branching lineage of eupolypods I, and have shown that this genus is not monospecific, but the exact species diversity is unknown. In this study, a new phylogeny is reconstructed based on an expanded taxon sampling and six molecular markers. Our major results include: (i) Didymochlaena is moderately or weakly supported as sister to the rest of eupolypods I, highlighting the difficulty in resolving the relationships of this important fern lineage in the polypods; (ii) species in Didymochlaena are resolved into a New World clade and an Old World clade, and the latter further into an African clade and an Asian-Pacific clade; (iii) an unusual tripling of molecular, morphological and geographical differentiation in Didymochlaena is detected, suggesting single vicariance or dispersal events in individual regions and no evidence for reversals at all, followed by allopatric speciation at more or less homogeneous rates; (iv) evolution of 18 morphological characters is inferred and two morphological synapomorphies defining the family are recognized-the elliptical sori and fewer than 10 sori per pinnule, the latter never having been suggested before; (v) based on morphological and molecular variation, 22 species in the genus are recognized contrasting with earlier estimates of between one and a few; and (vi) our biogeographical analysis suggests an origin for Didymochlaena in the latest Jurassic-earliest Cretaceous and the initial diversification of the extant lineages in the Miocene-all but one species diverged from their sisters within the last 27 Myr, in most cases associated with allopatric speciation owing to geologic and climatic events, or dispersal.

Species boundaries within the recently diverged South American monocot‐like Eryngium (Apiaceae, Saniculoideae) based on biogeographic, climatic, morphological, and molecular data: E. cerradense, a new species from Paraguay

AbstractThe species of Eryngium with a monocot‐like habit are among the most taxonomically complex. They show low phenotypic and molecular differentiation due to their recent, rapid, and reticulate speciation during the Quaternary period. In this study, we evaluate the boundaries of the monocot‐like Eryngium regnellii and a closely related entity from the grasslands in the Cerrado of Paraguay. We integrated evidence from multiple data sources to test species delimitation: we estimated the phylogenetic position of both entities within Eryngium based on ITS nrDNA and cpDNA sequences; we carried out univariate and multivariate morphometric analyses to test statistically significant differences and to recognize morphological diagnostic characters; we also analyzed geographic distribution, habitat, and bioclimatic variables to evaluate divergence in environmental niche, and finally, we used Bayesian coalescent‐based delimitation approaches to test genetic structure. Divergence in morphology, environmental niche, and genetic structure between E. regnellii and the lineage from Paraguay support the hypothesis of evolutionary independence among these two lineages. Therefore, based on an integrative approach, we recognize and describe a new species from Paraguay: Eryngium cerradense.

Carexquixotiana (Cyperaceae), a new Iberian endemic from Don Quixote's land (La Mancha, S Spain).

Despite centuries of work, the basic taxonomic knowledge of the flora of the Iberian Peninsula is still incomplete, especially for highly diverse and/or difficult genera such as Carex. In this study, we conducted an integrative systematic study based on molecular, morphological and cytogenetic data to elucidate the taxonomic status of several problematic Carex populations from La Mancha region (S Spain) belonging to Carexsect.Phacocystis. These populations have been traditionally considered of uncertain taxonomic adscription, but close to C.reuteriana due to their morphological appearance and ecological preferences. A detailed morphological and cytogenetic study was performed on 16 La Mancha's problematic populations (Sierra Madrona and Montes de Toledo) to compare them with the other Iberian sect. Phacocystis species. In addition, a phylogenetic analysis was conducted using two nuclear (ITS, ETS) and two plastid (rpl32-trnLUAG, ycf6-psbM) DNA regions, including representatives from all species of sect. Phacocystis. We found a significant degree of molecular and morphological differentiation that supports the recognition of La Mancha's problematic populations as a new Iberian endemic species, described here as Carexquixotiana Ben.Benítez, Martín-Bravo, Luceño & Jim.Mejías. Our results reveal that C.quixotiana, unexpectedly, is more closely related to C.nigra than to C.reuteriana on the basis of phylogenetic relationships and chromosome number. These contrasting patterns reflect the taxonomic complexity in sect. Phacocystis and highlight the need for integrative systematic approaches to disentangle such complicated evolutionary scenarios.

The Group Contribution to the Function Derived from Density and Speed-of-Sound Measurements for Glymes in N,N-Dimethylformamide + Water Mixtures.

The density and speed of sound of pentaglyme and hexaglyme in the N,N-dimethylformamide + water mixture at four temperatures are presented. The limiting apparent molar volumes (VΦ,m0=Vm0), the isobaric molar thermal expansion (Ep,m0), the isentropic compressibility (κS), and the limiting partial molar isentropic compression (KS,Φ,m0 = KS,m0) were calculated. Changes in the values obtained from the physicochemical parameters, as functions of composition and temperature, were analyzed in terms of the molecular interactions and structural differentiation of the investigated systems. The hydrophobic hydration process of the studied glymes was visible in the area of high water content in the mixture. The hydration number of glymes in water at four temperatures was calculated and analyzed. The contribution of the -CH2- and -O- group to the functions describing the volume and acoustic properties of the investigated system was calculated. The calculated values of the functions analyzed using the group contribution are in agreement with the values obtained from the experimental data. Thus, such contributions are valuable for wide ranges of data, which can be used to analyze the hydrophobic hydration and preferential solvation processes, as well as to calculate the values of these functions for other similar compounds.

Mass spectrometry in the molecular differentiation between SARS-CoV-2 Spike glycoprotein and mRNA anti COVID-19 vaccine Spike glycoprotein.

Mass spectrometry in the molecular differentiation between SARS-CoV-2 Spike glycoprotein and mRNA anti COVID-19 vaccine Spike glycoprotein.

Genetic polymorphism detection in brazilian perennial cottons (Gossypium spp.) using an ISSR marker system and its application for molecular interspecific differentiation.

The semi-domesticated Brazilian perennial cotton (Gossypium spp.) germplasm is considered a source of variability for creating modern upland cotton varieties. Here we used Inter-simple Sequence Repeat (ISSR) markers to detect intra and interspecific genetic polymorphism in Gossypium hirsutum L. r. marie-galante and Gossypium barbadense L. and to use molecular data to assessing genetic diversity and molecular discrimination of these species. The sets contained 12 G. barbadense genotypes and 16 G. hirsutum genotypes from a Brazilian collection. The 11 ISSR primers were used for genotyping yielded 101 bands (polymorphism = 47.5%) and were classified as moderately informative (PIC = 0.304). The ISSR markers exposed a greater diversity in G. hirsutum (P = 24.72%; HE =0.071 and I = 0.111) as compared to G. barbadense (P = 17.98%, HE = 0.043 and I = 0.070). The AMOVA analysis showed that 89.47% of the genetic variation was partitioned within species which is supported by Nei's genetic differentiation (Gst = 0.598) and gene flow (Nm = 0.338), suggesting that strong reproductive barriers between species. The UPGMA Cluster Analysis, Principal Coordinate Analysis and Bayesian Model-Based Structural Analysis divided the 28 genotypes into two main clades consistent with the taxonomical delimitation. The ISSR marker system offers a new approach to determining molecular differences between two cotton species (G. hirsutum L. r. marie-galante and G. barbadense L.). This study can expand the molecular marker resources for the identification and improvement of our knowledge about the genetic diversity and relationships between perennial cotton genotypes.

Context-dependent gene regulatory network reveals regulation dynamics and cell trajectories using unspliced transcripts.

Gene regulatory networks govern complex gene expression programs in various biological phenomena, including embryonic development, cell fate decisions and oncogenesis. Single-cell techniques are increasingly being used to study gene expression, providing higher resolution than traditional approaches. However, inferring a comprehensive gene regulatory network across different cell types remains a challenge. Here, we propose to construct context-dependent gene regulatory networks (CDGRNs) from single-cell RNA sequencing data utilizing both spliced and unspliced transcript expression levels. A gene regulatory network is decomposed into subnetworks corresponding to different transcriptomic contexts. Each subnetwork comprises the consensus active regulation pairs of transcription factors and their target genes shared by a group of cells, inferred by a Gaussian mixture model. We find that the union of gene regulation pairs in all contexts is sufficient to reconstruct differentiation trajectories. Functions specific to the cell cycle, cell differentiation or tissue-specific functions are enriched throughout the developmental process in each context. Surprisingly, we also observe that the network entropy of CDGRNs decreases along differentiation trajectories, indicating directionality in differentiation. Overall, CDGRN allows us to establish the connection between gene regulation at the molecular level and cell differentiation at the macroscopic level.