Sequence Determination Research Articles

Affinity Maturated Transferrin Receptor Apical Domain Blocks Machupo Virus Glycoprotein Binding

Transferrin receptor 1 (TfR) delivers iron across cellular membranes by shuttling the ion carrier protein transferrin. This ability to deliver large protein ligands inside cells is taken advantage of by pathogens to infiltrate human cells. Notably, the receptor’s outermost ectodomain, the apical domain, is used as a point of attachment for several viruses including hemorrhagic arenaviruses. To better understand interactions with the receptor it would be advantageous to probe sequence determinants in the apical domain with viral spike proteins. Here, we carried out affinity maturation of our computationally designed apical domain from human TfR to identify underlying driving forces that lead to better binding. The improved variants were confirmed by in vitro surface plasmon resonance measurements with dissociation constants obtained in the lower nanomolar range. It was found that the strong binding affinities for the optimized variants matched the strength of interactions with the native receptor. The structure of the best variant was determined experimentally indicating that the conformational change in the hairpin binding motif at the protein–protein interface plays a crucial role. The experimental methodology can be straightforwardly applied to other arenavirus or pathogens that use the apical domain. It can further be useful to probe host–virus compatibility or therapeutic strategies based on the transferrin receptor decoys.

A quantitative analysis of spontaneous alternation behaviors on a Y-maze reveals adverse effects of acute social isolation on spatial working memory

Animals tend to alternate between different choices, which requires the ability to remember recent choices. The Y-maze spontaneous alternation test is widely used in various animal models for assessing short-term memory, and its precise evaluation depends upon the accurate determination of the arm visit sequence. However, an objective method for defining arm visits is lacking owing to uncertainty regarding the extent to which an animal must go into the arm to be considered visited. Here, we conducted quantitative analyses on mice behavior in the Y-maze while systematically varying the arm visit threshold and assessed the effect of acute social isolation on spatial working memory. Our results revealed that 24-h social isolation significantly reduced spontaneous alternation rate when the arm threshold was set at the distal part of the arm. Furthermore, the memory of the recently visited arms faded away faster in the socially isolated mice. However, other behavioral factors were comparable to those of the group-housed mice, indicating a specific impairment of short-term memory. Our findings suggest that the location of arm visit threshold is critical for the precise evaluation of short-term memory, and our study provides a method for comprehensively and systematically assessing spontaneous alternation behavior in the Y-maze.

Multimodal learning of noncoding variant effects using genome sequence and chromatin structure.

A growing amount of noncoding genetic variants, including single-nucleotide polymorphisms, are found to be associated with complex human traits and diseases. Their mechanistic interpretation is relatively limited and can use the help from computational prediction of their effects on epigenetic profiles. However, current models often focus on local, 1D genome sequence determinants and disregard global, 3D chromatin structure that critically affects epigenetic events. We find that noncoding variants of unexpected high similarity in epigenetic profiles, with regards to their relatively low similarity in local sequences, can be largely attributed to their proximity in chromatin structure. Accordingly, we have developed a multimodal deep learning scheme that incorporates both data of 1D genome sequence and 3D chromatin structure for predicting noncoding variant effects. Specifically, we have integrated convolutional and recurrent neural networks for sequence embedding and graph neural networks for structure embedding despite the resolution gap between the two types of data, while utilizing recent DNA language models. Numerical results show that our models outperform competing sequence-only models in predicting epigenetic profiles and their use of long-range interactions complement sequence-only models in extracting regulatory motifs. They prove to be excellent predictors for noncoding variant effects in gene expression and pathogenicity, whether in unsupervised "zero-shot" learning or supervised "few-shot" learning. Codes and data can be accessed at https://github.com/Shen-Lab/ncVarPred-1D3D and https://zenodo.org/record/7975777.

Studies on the complex Warnowiaceae (Dinophyceae) I. Lohmann’s Pouchetia parva refound and renamed Nematodinium parvum comb. nov. (= Warnowia parva)

ABSTRACT The dinoflagellate family Warnowiaceae has often been considered to include some of the most complex cells among the protists. The number of described species is around 40, but both the species and generic concepts are in need of revision. Warnowiaceans are often fragile and readily change morphology under the light microscope, and they are usually regarded to be rare. They are particularly famous for the eye-like structures, termed ocelloids. Studies on warnowiaceans are hampered by lack of cultures, and our studies are therefore based on cells obtained directly from field samples. We provide a description of Nematodinium parvum comb. nov. (syn. Pouchetia parva, Warnowia parva), based on light, scanning and transmission electron microscopy, combined with phylogenetic analyses of ribosomal genes. It was described in 1908 by Lohmann from Kieler Bay, but is often common in Danish waters, allowing observations on distribution and behaviour. Crucial conditions for finding high cell abundances were periods of warm temperatures and a calm sea. Cells were yellowish, photosynthetic, and contained a net-like chloroplast, in addition to an ocelloid, trichocysts and harpoon-like nematocysts. They divided asexually but planozygotes were also seen. Following the demonstration of nematocysts, the species, which has been known as Warnowia parva since 1928, is transferred to Nematodinium. The finding of a peduncle indicates mixotrophy but all feeding experiments failed to identify a suitable prey. The phylogenetic analyses based on single-cell PCR and sequence determinations of small and large subunit rDNA confirmed that the systematics of Nematodinium and Warnowia is in a state of flux.

Surveillance of human Group A rotavirus in Ningxia, China (2015-2021): Emergence and prevalence of G9P[8]-E2 and G3P[8]-E2 genotypes.

Group A rotaviruses (RVA) are the primary pathogens of acute gastroenteritis. Currently, two live attenuated RVA vaccines, LLR and RotaTeq, have been introduced into mainland China but are not included in the national immunization program. Because of the unknown genetic evolution of group A rotavirus in an all-age population in Ningxia, China, we monitored the epidemiological characteristics and circulating genotypes of RVA as a reference for developing vaccine strategies. We conducted seven years of consecutive surveillance of RVA based on stool samples from patients with acute gastroenteritis in sentinel hospitals in Ningxia, China, from 2015 to 2021. Reverse transcription quantitative polymerase chain reaction(RT-qPCR) was used to detect RVA in stool samples. Genotyping and phylogenetic analysis of VP7, VP4 and NSP4 genes were performed by reverse transcription-polymerase chain reaction(RT-PCR) and nucleotide sequence determination. RVA was detected in 16.58% (1436/8662) of 8662 stool samples. The positive rates were 7.17% (201/2805) and 21.09% (1235/5857) in adults and children, respectively. The most affected age group was infants and children aged 12-23months, with a positive rate of 29.53% (p<0.05). A significant winter/spring seasonality was observed. 23.29% positive rate in 2020 was the highest in 7years (p<0.05). The region with the highest positive rate in the adult group was Yinchuan, and the children's group was Guyuan. A total of 9 genotype combinations were found to be distributed in Ningxia. The dominant genotype combinations in this region gradually changed from G9P[8]-E1, G3P[8]-E1, G1P[8]-E1 to G9P[8]-E1, G9P[8]-E2, and G3P[8]-E2 during these seven years. Rare strains (e.g., G9P[4]-E1, G3P[9]-E3 and G1P[8]-E2) were occasionally detected during the study. During the study period, changes in the significant RVA circulating genotype combinations and the emergence of reassortment strains were observed, particularly the emergence and prevalence of G9P[8]-E2, G3P[8]-E2 reassortants in the region. These results indicate the importance of continuous monitoring of the molecular evolution and recombination characteristics of RVA, and should not be limited to G/P genotyping but should consider multi-gene fragment co-analysis and whole genome sequencing.

Peptides From the Variable Domain of Immunoglobulin G as Biomarkers in Chronic Inflammatory Demyelinating Polyradiculoneuropathy.

Chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) is a clinically heterogeneous immune-mediated disease. Diagnostic biomarkers for CIDP are currently lacking. Peptides derived from the variable domain of circulating immunoglobulin G (IgG) have earlier been shown to be shared among patients with the same immunologic disease. Because humoral immune factors are hypothesized to be involved in the pathogenesis of CIDP, we evaluated IgG variable domain-derived peptides as diagnostic biomarkers in CIDP (primary objective) and whether IgG-derived peptides could cluster objective clinical entities in CIDP (secondary objective). IgG-derived peptides were determined in prospectively collected sera of patients with CIDP and neurologic controls by means of mass spectrometry. Peptides of interest were selected through statistical analysis in a discovery cohort followed by sequence determination and confirmation. Diagnostic performance was evaluated for individual selected peptides and for a multipeptide model incorporating selected peptides, followed by performance reassessment in a validation cohort. Clustering of patients with CIDP based on IgG-derived peptides was evaluated through unsupervised sparse principal component analysis followed by k-means clustering. Sixteen peptides originating from the IgG variable domain were selected as candidate biomarkers in a discovery cohort of 44 patients with CIDP and 29 neurologic controls. For all 16 peptides, univariate logistic regressions and ROC curve analysis demonstrated increasing peptide abundances to associate with increased odds for CIDP (area under the curves [AUCs] ranging from 64.6% to 79.6%). When including age and sex in the logistic regression models, this remained the case for 13/16 peptides. A model composed of 5/16 selected peptides showed strong discriminating performance between patients with CIDP and controls (AUC 91.5%; 95% CI 84.6%-98.4%; p < 0.001). In the validation cohort containing 45 patients and 43 controls, 2/16 peptides demonstrated increasing abundances to associate with increased odds for CIDP, while the five-peptide model demonstrated an AUC of 61.2% (95% CI 49.3%-73.2%; p = 0.064). Peptide-based patient clusters did not associate with clinical features. IgG variable domain-derived peptides showed a valid source for diagnostic biomarkers in CIDP, albeit with challenges toward replication. Our proof-of-concept findings warrant further study of IgG-derived peptides as biomarkers in more homogeneous cohorts of patients with CIDP and controls. This study provides Class III evidence that the pattern of serum IgG-derived peptide clusters may help differentiate between patients with CIDP and those with other peripheral neuropathies.

Integrated Transcriptome and Biochemical Analysis Provides New Insights into the Leaf Color Change in Acer fabri

Acer fabri is a widely distributed ornamental tree with colorful leaves and high ornamental value. Its young leaves change from red to red and green until turning fully green. To understand the mechanism of leaf color change, transcriptome sequencing and pigment content determination were performed in three stages during the leaf color change of A. fabri. In total, 53,550 genes, including 838 transcription factors (TFs), were identified by transcriptome sequencing. In addition, the results of orthogonal partial least squares-discriminant analysis (OPLS-DA) of three pigments in the three stages of leaf color development suggested that carotenoids played a major role in the process of leaf color change from red to red-green, whereas anthocyanins played an important role in the process of leaf color change from red to green. Based on weighted gene co-expression network analysis (WGCNA), Af0034384 (HSFB2A), Af0051627 (NMT1), and Af0052541 (THY-1) were selected as hub genes from characteristic modules with significant correlation between carotenoids and anthocyanins. The results of gene network regulation maps and real-time fluorescence quantitative PCR (qRT-PCR) showed that Af0010511 (NAC100) upregulated the expression of Af0034384 (HSFB2A), leading to an increase in carotenoid content and the gradual greening of leaves during the transition from red to green. However, during the transition from red to green leaves, Af0033232 (NAC83) and Af0049421 (WRKY24) downregulated the expression of Af0051627 (NMT1) and Af0052541 (THY-1), respectively, leading to a decrease in anthocyanin content and the complete greening of leaves. These results could provide new ideas for studying the molecular mechanism of leaf color change in A. fabri and other species.

Molecular epidemiological study on rubella virus circulating in Yunnan Province during 2011-2021

Objective: To understand the genotype distribution and transmission pattern of rubella virus (RuV) circulating in Yunnan Province. Methods: Throat swab samples were collected from rubella outbreaks and sporadic cases in nine prefectures/cities of Yunnan Province from 2011 to 2021. Virus isolation, amplification of target genes and sequence determination were performed on the RuV-positive samples. The genotypes and lineages of Yunnan strains were determined by comparing them with the reference strains, and further phylogenetic analysis was performed with Yunnan strains and strains circulating in other provinces of China during the same period. Results: RuV circulating in Yunnan province during 2011-2021 showed significant genetic diversity, and three lineages, 1E-L1, 2B-L1 and 1E-L2, were detected. Two lineage-switches were also identified, including the conversion of 1E-L1 to 2B-L1 between 2012 and 2013, and the replacement of 2B-L1 to 1E-L2 after 2018. The time of the switches was basically consistent with the outbreak in Yunnan province in 2012 and the time of the rubella reemergence and epidemic between 2018 and 2019. The amino acid sequence of RuV virus strains in Yunnan province was highly conserved, and no important functional regions were changed. Conclusions: The transmission pattern of RuV in Yunnan province is generally consistent with the epidemic trend of RuV in other provinces of China.

A multiplexed bacterial two-hybrid for rapid characterization of protein–protein interactions and iterative protein design

Protein-protein interactions (PPIs) are crucial for biological functions and have applications ranging from drug design to synthetic cell circuits. Coiled-coils have been used as a model to study the sequence determinants of specificity. However, building well-behaved sets of orthogonal pairs of coiled-coils remains challenging due to inaccurate predictions of orthogonality and difficulties in testing at scale. To address this, we develop the next-generation bacterial two-hybrid (NGB2H) method, which allows for the rapid exploration of interactions of programmed protein libraries in a quantitative and scalable way using next-generation sequencing readout. We design, build, and test large sets of orthogonal synthetic coiled-coils, assayed over 8,000 PPIs, and used the dataset to train a more accurate coiled-coil scoring algorithm (iCipa). After characterizing nearly 18,000 new PPIs, we identify to the best of our knowledge the largest set of orthogonal coiled-coils to date, with fifteen on-target interactions. Our approach provides a powerful tool for the design of orthogonal PPIs.

Self-expansion full information optimization strategy: Convenient and efficient method for near infrared spectrum auto-analysis

An essential step in the application of near infrared spectroscopy technology is the spectrum preprocessing. A reasonable implementation of it ensures that the effective spectral information is correctly extracted and, also that the model's accuracy is increased. However, some analysts' research still uses the manual approach of trial and error, particularly those less skilled ones. Previous papers have provided preprocessing optimization algorithms for NIR, but there are still some problems that need to be resolved, such as the unwieldy sequence determination of preprocessing method or, the fluctuated optimization outcomes or, lack of sufficient statistical information. This research suggests a spectrum auto-analysis methodology named self-expansion full information optimization strategy, a new powerful open-source technique for concurrently addressing all of these above issues simultaneously. For the first time in the field of chemometrics, this algorithm offers a reliable and effective automatic near infrared auto-modelling method based on the statistical informatics. With the aid of its built-in modules, such as information generators, spectrum processors, etc., it is able to fully search the common preprocessing techniques, which is determined by Monte Carlo cross validation. Then the final ensemble calibration model is built by employing the optimized preprocessing schemes, along with the wavelength variables screening algorithm. The optimization strategy can offer the user objective useful statistics information created throughout the modeling process to further examine the model's effectiveness. The results demonstrate that the suggested method can easily and successfully extract spectrum information and develop calibration models by putting it to the test on two groups of actual near-infrared spectral data. Additionally, this optimization strategy can also be applied to other spectrum analysis areas, such Raman spectroscopy or infrared spectroscopy, by changing a few of its parameters, and has extraordinary application value.

Determination of Whole Genome Sequence and Resistance Genes in Hypermucoviscous Klebsiella pneumoniae with Long-Read Sequencing Technology

Numerous virulence factors are present in the hypervirulent/hypermucoviscous Klebsiella pneumoniae, which primarily causes community-acquired infections. In recent years, hypervirulent/hypermucoviscous K.pneumoniae has acquired resistance genes and has been linked to healthcare-associated infections. The aims of this study were to determine whole genome sequencing of hypermucoviscous K.pneumoniae that induces healthcare-associated bloodstream infection utilizing the Oxford Nanopore (MinION) platform, to identify resistance genes using various databases, and to compare the database results. K.pneumoniae isolates that were sent to the Dokuz Eylül University Research and Training Hospital Center Laboratory and were isolated from blood cultures were included in this study between January 2018 and December 2020. K.pneumoniae isolates were identified using the automated VITEK-2 system. The disc diffusion method was used to characterize the antimicrobial resistance profile, and the string test was used to assess the hypermucoviscous phenotype. By using specific primers blaOXA-48, blaNDM, blaKPC, blaIMP, and blaVIM, carbapenem resistance genes were investigated using the PCR method. To ascertain clonal relatedness among hypermucoviscous K.pneumoniae isolates, PFGE was used. The whole genome sequencing of five K.pneumoniae strains with different origins was determined by Oxford Nanopore (MinION) technology. Using the ResFinder, CARD and BacWGSTdb databases, resistance genes were examined. Capsule regulation genes were analyzed with the BacWGSTdb database. Resistance genes on the plasmid were discovered using the ResFinder database after plasmid analyses were carried out using the PLSDB and PlasmidFinder databases. A total of 244 K.pneumoniae isolates were included in the study. Ten isolates were found to be hypermucoviscous. A carbapenem-resistant hypermucoviscous K.pneumoniae isolate was discovered to carry the blaOXA-48. Five hypermucoviscous isolates of which whole genome sequences were determined had blaSHV types, oqxB, oqxA, and fosA genes on the chromosome. Capsule regulation genes rcsA, rcsB were detected in all five isolates, and rmpA/rmpA2 genes caused hypermucoviscous phenotype was detected in two of the five isolates. In the plasmid analysis, IncFIB (K) pCAV1099- 14, Col (pHAD28), IncFIA (HI1)-IncR, Col (pHAD28), Col440, IncFIB (pNDM_Mar)-IncHI1 (pNDM_MAR), IncL, IncFIB (K)-IncHI1 (K), IncFIB (K), IncR, IncFIB (pNDM_Mar)-IncHI1 (pNDM_MAR)-IncR, repB plasmids were identified. Resistance genes; aac(3)-IId, aph(6)-Id, aph(3")-Ib qnrB1, sul2, dfrA14, blaTEM-1A, blaCTX-M-15, armA, msr(E), mph(E), catB3, blaOXA-1, aac(6')-Ib-cr, catA1 and blaOXA-48 were detected on the plasmid. In the bioinformatic analyzes, it was determined that two study isolates with hypermucoviscous phenotype had various plasmids and carried many resistance genes on these plasmids. Various resistance and virulence genes are spread through plasmids and the number of resistant hypermucoviscous K.pneumoniae is increasing day by day. Appropriate infection control strategies should be implemented to prevent healthcare-associated infections and outbreaks caused by antimicrobial-resistant hypermucoviscous K.pneumoniae. For this purpose, resistance monitoring of antimicrobials used in the treatment of K.pneumoniae infections should be performed. There were some differences in the results of the three databases used to detect resistance genes in this study. For this reason, it is important to analyze different databases and compare the database results in studies while performing whole genome sequencing. Further studies and updates using different databases will increase the compatibility between the databases.

Evolutionary multitasking for bidirectional adaptive codec: A case study on vehicle routing problem with time windows

The vehicle routing problem (VRP), an NP-hard problem of considerable complexity, pertains to the determination of optimal sequences of customer visits in an effort to minimize the total operational cost of vehicles. Conventional approaches to tackling VRP have primarily focused on optimizing each task independently, utilizing mathematical methodologies or evolutionary algorithms. Nevertheless, research exploring the application of the advanced concept of evolutionary multitasking (EMT) to address VRP remains limited. To enrich the body of knowledge in this field, our study presents an innovative EMT algorithm, dubbed EMT-RD. This algorithm employs a bidirectional adaptive codec to facilitate the transfer of information between real and discrete solutions. The EMT-RD algorithm permits the transfer of real and discrete solutions across varying tasks, utilizing an encoding rule that transforms information from real to discrete solutions for each task. Further, a decoding rule from discrete to real solutions generates search experiences, facilitating the transfer of knowledge between real solutions. The mechanism is capped off with an adaptive transfer strategy that instigates transfer operations across different tasks. Each task is tackled using a single-task optimization algorithm that employs an artificial bee colony algorithm. Through comprehensive experimentation, we found that our proposed EMT-RD algorithm outperforms competitors on 168 VRP with time windows multitasking instance pairs and four real-world problem pairs that were developed for this study. These results underscore the efficacy of the adaptive transfer strategy and bidirectional adaptive codec in addressing inter-task correlations when solving the VRP.

Some structural features of the peptide profile of myelin basic protein-hydrolyzing antibodies in schizophrenic patients.

The antibodies of schizophrenic patients that hydrolyze myelin basic protein (MBP) have been actively studied recently, but the mechanism of the catalytic properties of immunoglobulin molecules remains unknown. Determination of specific immunoglobulin sequences associated with the high activity of MBP proteolysis will help to understand the mechanisms of abzyme catalysis. In the course of comparative mass spectrometric analysis of IgG peptides from the blood serum of patients with acute schizophrenia and healthy people, 12 sequences were identified, which were found only in antibodies that hydrolyze MBP. These sequences belong to IgG heavy chains and κ- and λ-type light chains, with eight of them belonging to variable domains. The content of peptides from the variable regions of the light chains does not correlate with the proteolytic activity of IgG to MBP in patients with schizophrenia, whereas for two sequences from the variable regions of the heavy chains (FQ(+0.98)GWVTMTR and *LYLQMN(+0.98)SLR), an increase in activity with increasing their concentration. The results suggest that these sequences may be involved in one way or another in MBP hydrolysis.

Combining TSS-MPRA and sensitive TSS profile dissimilarity scoring to study the sequence determinants of transcription initiation.

Cis-regulatory elements (CREs) can be classified by the shapes of their transcription start site (TSS) profiles, which are indicative of distinct regulatory mechanisms. Massively parallel reporter assays (MPRAs) are increasingly being used to study CRE regulatory mechanisms, yet the degree to which MPRAs replicate individual endogenous TSS profiles has not been determined. Here, we present a new low-input MPRA protocol (TSS-MPRA) that enables measuring TSS profiles of episomal reporters as well as after lentiviral reporter chromatinization. To sensitively compare MPRA and endogenous TSS profiles, we developed a novel dissimilarity scoring algorithm (WIP score) that outperforms the frequently used earth mover's distance on experimental data. Using TSS-MPRA and WIP scoring on 500 unique reporter inserts, we found that short (153 bp) MPRA promoter inserts replicate the endogenous TSS patterns of ∼60% of promoters. Lentiviral reporter chromatinization did not improve fidelity of TSS-MPRA initiation patterns, and increasing insert size frequently led to activation of extraneous TSS in the MPRA that are not active in vivo. We discuss the implications of our findings, which highlight important caveats when using MPRAs to study transcription mechanisms. Finally, we illustrate how TSS-MPRA and WIP scoring can provide novel insights into the impact of transcription factor motif mutations and genetic variants on TSS patterns and transcription levels.

Decolorization of p-nitrophenol and draft genome sequence of Pseudomonas sp. strain PNPG3: A preliminary report

Aim: To study decolorization (with concomitant depletion) of p-nitrophenol by a bacterial strain designated as PNPG3 and determination of draft genome sequence of the strain to understand its potential. Methodology: A comparative study of PNP’s decolorization (with concomitant removal) in three different test conditions was undertaken. The experiment was carried out in one-liter volume Schott Duran bottles. Genomic DNA was extracted and draft genome sequence was determined using Illumina HiSeqX platform. Raw reads were assembled and subjected to subsystem classification using several bioinformatics tools (RAST, PATRIC, and NCBI’s PGAP pipelines). The genome sequence was deposited at the NCBI Genome database and the strain PNPG3 was also deposited at MTCC, IMTECH, Chandigarh. Results: The bacterial strain PNPG3 could carry out decolorization with concomitant removal of PNP in all three sets of experiments, including one set, where only distilled water was used. The best decolorization (with concomitant PNP removal) capacity was recorded for set D (with Minimal Medium, MM; PNP, and free cells) followed by set E (MM, PNP, and immobilized cells) and set B (distilled water, PNP and free cells) conditions. The size of the draft genome sequence of the strain PNPG3 was 6,566,321 bp, with 62.26% GC contents. The genome had 6210 protein-coding sequences and 66 tRNA genes. The predictive presence of different types of proteases and siderophore receptors indicated its possible potential for industrial applications and plant growth-promoting activities. Interpretation: The bacterium Pseudomonas sp. strain PNPG3 has the capacity to decolorize p-nitrophenol even in presence of distilled water and it remains viable for up to twelve days. The genome sequence revealed that the strain harbored genes responsible for the metabolism of aromatic compounds, chemotaxis, protease, and siderophore receptors indicating the versatile nature of the strain. Key words: Decolorization, P-nitrophenol, Pseudomonas sp., RAST, Xenobiotic

Tectonic setting of Kaladgi-Badami basin and its possible connection with adjacent Proterozoic basins, Karnataka, India

Sedimentary rock types and basin geometries are interrelated with characteristic tectonic settings. In fact, sedimentation pattern at central stable cratonic areas are not same as that on continental margins. Kaladgi-Badami (KB) basinal feldspathic arenite (i.e., arkose) dominated rocks contain more plagioclase than potassium feldspar unlike the opposite scenario of Phanerozoic sandstones. Tectonic history of the KB basin has been interpreted since basin development, sedimentation through interrupting hiatus, tectonic reworking, redeposition at later developed depocenters and later exhumation, erosion, tectonism till Deccan volcanism and finally latest configuration. As per the International commission of Stratigraphy (ICS), principles of stratigraphic classification and usages of terminology of fundamental lithostratigraphic unit ‘Formation’ is reexamined in this paper. The definition of Formation as “smallest mappable rock unit with a definite lithologic characteristic that allow it to be distinguished from other such units” is improperly used because without mentioning a specific scale every unit is mappable or traceable. Therefore, the minimum mappable unit means the unit which can be at least visible in a scale in which entire basin can be seen. Based on this slight modification is proposed in the stratigraphy of KB basin. Geochronology based radiometric stratigraphy is best tool for determination of time sequence of geological events. However, it is always a matter of concern that dating methods and materials are often not suitable in sedimentary geology. In case of passive rifted sedimentary basins like Kaladgi, Badami and Bhima of the Dharwar Craton, there is negligible igneous events and indirect dating methods are mostly available. Detrital zircon dating can give maximum age or provenance age, which cannot be of much use. However, there are other indirect tools, which are already utilized by several workers earlier and based on the reviews and present observation and mapping compilation it is proposed that KB and Bhima basins used to be a single basin, and later tectonism followed by Deccan volcanism affected the present geographic continuity. Since the distance between westernmost Bhima basinal rocks and easternmost KB basinal rocks near Mudhebihal is nearly around 15km, it is logical to consider that these were geographically connected, because there is an intense faulting near to these basin margins.

Molecular authentication of some rare Iris (Iridaceae) species from Uzbekistan

The determination of nucleotide sequences in DNA, sequencing of chloroplast genomes techniques, based on the use of combinations a significant number genetic markers, as well as the nuclear genome are widely use for inventory of rare plant species, ecological monitoring and species diversity assessment. The results of our molecular analysis are used to examine the taxonomic identity and phylogenetic relationship of the studied Iris species collected in Uzbekistan. The genotyping of three-four functional loci of nrDNA and cpDNA was performed for efficient species identification and confident results. The nuclear ITS gene sequences and chloroplast trnL-trnF, rbcL, matK gene sequences were obtained from 10 Iris species and have proven to be useful as molecular markers for species identification. Sequences of cpDNA regions, have been used to assess interspecific relationships of Iris. Hierarchical clustering were constructed using 13 consensus matK gene sequences. In this study, we consider the possibility of successful application the DNA barcoding as a tool for assessment of wild Iris species diversity in Uzbekistan.

Hybrid-seq deciphers the complex transcriptional profile of the human BRCA1 DNA repair associated gene

ABSTRACT Breast Cancer Gene 1 (BRCA1) is a tumour suppressor protein that modulates multiple biological processes including genomic stability and DNA damage repair. Although the main BRCA1 protein is well characterized, further proteomics studies have already identified additional BRCA1 isoforms with lower molecular weights. However, the accurate nucleotide sequence determination of their corresponding mRNAs is still a barrier, mainly due to the increased mRNA length of BRCA1 (~5.5 kb) and the limitations of the already implemented sequencing approaches. In the present study, we designed and employed a multiplexed hybrid sequencing approach (Hybrid-seq), based on nanopore and semi-conductor sequencing, aiming to detect BRCA1 alternative transcripts in a panel of human cancer and non-cancerous cell lines. The implementation of the described Hybrid-seq approach led to the generation of highly accurate long sequencing reads that enabled the identification of a wide spectrum of BRCA1 splice variants (BRCA1 sv.7 – sv.52), thus deciphering the transcriptional landscape of the human BRCA1 gene. In addition, demultiplexing of the sequencing data unveiled the expression profile and abundance of the described BRCA1 mRNAs in breast, ovarian, prostate, colorectal, lung and brain cancer as well as in non-cancerous human cell lines. Finally, in silico analysis supports that multiple detected mRNAs harbour open reading frames, being highly expected to encode putative protein isoforms with conserved domains, thus providing new insights into the complex roles of BRCA1 in genomic stability and DNA damage repair.

Y225A induces long-range conformational changes in human prion protein that are protective in Drosophila

Prion protein (PrP) misfolding is the key trigger in the devastating prion diseases. Yet the sequence and structural determinants of PrP conformation and toxicity are not known in detail. Here, we describe the impact of replacing Y225 in human PrP with A225 from rabbit PrP, an animal highly resistant to prion diseases. We first examined human PrP-Y225A by molecular dynamics simulations. We next introduced human PrP in Drosophila and compared the toxicity of human PrP-WT and Y225A in the eye and in brain neurons. Y225A stabilizes the β2-α2 loop into a 310-helix from six different conformations identified in WT and lowers hydrophobic exposure. Transgenic flies expressing PrP-Y225A exhibit less toxicity in the eye and in brain neurons and less accumulation of insoluble PrP. Overall, we determined that Y225A lowers toxicity in Drosophila assays by promoting a structured loop conformation that increases the stability of the globular domain. These findings are significant because they shed light on the key role of distal α-helix 3 on the dynamics of the loop and the entire globular domain.

Evaluation of adiponectin and TNF-α expression in diabetic patients and its relationship with cardiovascular diseases.

Diabetes is caused by peripheral insulin resistance and lack of insulin secretion due to the apoptosis of pancreatic beta cells. Tumor necrosis factor alpha (TNF-α), a pro-inflammatory cytokine secreted from the tissue on the insulin signaling pathway, can play a role in causing fat resistance to insulin in type 2 diabetes patients. Adiponectin is a specific protein of adipose tissue. It belongs to the collectin family, which is present in human plasma at a high level and can protect against vascular lesions. Considering the importance of epigenetic changes in the development of multifactorial diseases, this study was conducted to investigate the methylation of TNF-α gene promoter in patients with type diabetes with cardiovascular disease and compare it with diabetic people without cardiovascular disease. Also, the serum concentration of adiponectin was investigated in diabetic patients with and without cardiovascular disease. For this purpose, 95 patients with type 2 diabetes referred to Isfahan Endocrine and Metabolism Research Center were divided into two groups: cardiovascular disease and without cardiovascular disease, based on the angiography results. Serum adiponectin level was measured by the RIA method. In addition to adiponectin, indicators such as FBS, cholesterol, triglycerides, and HDL were also measured in these patients. Then, the promoter region of the TNF-α gene was investigated by bisulfite treatment method, nested PCR, and finally, sequence determination. The results showed that the serum level of adiponectin was higher in diabetic patients without cardiovascular disease than in diabetic patients with cardiovascular disease, but this difference was not statistically significant. Also, no change was observed between men and women in TNF-α gene promoter methylation in diabetic and non-diabetic groups. In general, the decrease in adiponectin concentration in diabetic people can be a factor in causing macroangiopathy, so it can be predicted that the production of recombinant adiponectin can be helpful in the treatment and protection of cardiovascular disease in these patients. Also, it seems that the epigenetic changes of cytokines that play a role in causing insulin resistance in type 2 diabetic patients are not noticeable in the peripheral blood sample. In this regard, other tissues should probably be investigated.