Stalling of Transcription by Putative G-quadruplex Sequences and CRISPR-dCas9.

Putative G-quadruplex forming sequences (PQS) have been identified in promoter sequences of prominent genes that are implicated among others in cancer and neurological disorders. We explored mechanistic aspects of CRISPR-dCas9-mediated gene expression regulation, which is transient and sequence specific unlike alternative approaches that lack such specificity or create permanent mutations, using the PQS in tyrosine hydroxylase (TH) and c-Myc promoters as model systems. We performed in vitro ensemble and single molecule investigations to study whether G-quadruplex (GQ) structures or dCas9 impede T7 RNA polymerase (RNAP) elongation process and whether orientation of these factors is significant. Our results demonstrate that dCas9 is more likely to block RNAP progression when the non-template strand is targeted. While the GQ in TH promoter was effectively destabilized when the dCas9 target site partially overlapped with the PQS, the c-Myc GQ remained folded and stalled RNAP elongation. We also determined that a minimum separation between the transcription start site and the dCas9 target site is required for effective stalling of RNAP by dCas9. Our study provides significant insights about the factors that impact dCas9-mediated transcription regulation when dCas9 targets the vicinity of sequences that form secondary structures and provides practical guidelines for designing guide RNA sequences.

The Cby3/ciBAR1 complex positions the annulus along the sperm flagellum during spermiogenesis.

Proper compartmentalization of the sperm flagellum is essential for fertility. The annulus is a septin-based ring that demarcates the midpiece (MP) and the principal piece (PP). It is assembled at the flagellar base, migrates caudally, and halts upon arriving at the PP. However, the mechanisms governing annulus positioning remain unknown. We report that a Chibby3 (Cby3)/Cby1-interacting BAR domain-containing 1 (ciBAR1) complex is required for this process. Ablation of either gene in mice results in male fertility defects, caused by kinked sperm flagella with the annulus mispositioned in the PP. Cby3 and ciBAR1 interact and colocalize to the annulus near the curved membrane invagination at the flagellar pocket. In the absence of Cby3, periannular membranes appear to be deformed, allowing the annulus to migrate over the fibrous sheath into the PP. Collectively, our results suggest that the Cby3/ciBAR1 complex regulates local membrane properties to position the annulus at the MP/PP junction.

Social and facilitating influences in fintech user intention and the fintech gender gap.

Given the advances in technology, Fintech is an invaluable tool which allows unbanked people to access financial services when social, cultural, economic and technological factors affect their user intentions (UI). Despite the great importance of the role of social and facilitating influences in the adoption of Fintech services, little research has been conducted on how and what influences affect Fintech user intention (FUI) and whether there is a gender gap in FUI. Therefore, this study aims to help formulate effective Fintech policies and close the gender gap by investigating the role of social and facilitating influences and sociodemographic variables in FUI. The study sample comprised 237 participants, and the data were collected through interviews with the use of a structured questionnaire in Chattogram, Bangladesh. The collected data were analysed employing exploratory factor analysis and an ordered logistic regression model. The study also examined the Fintech gender gap by applying the Blinder Oaxaca decomposition model. The results reveal that image, compatibility and the experiences of Fintech use are the positive and significant predictors of FUI, with the perceived social norm for adopting Fintech being non-informative for users. There is a significant interaction between user compatibility and experience of use in relation to Fintech. Interestingly, perceived behavioural control negatively influenced females to adopt Fintech. Furthermore, the study found a gender gap in FUI. The findings have managerial implications.

CovTiNet: Covid text identification network using attention-based positional embedding feature fusion.

Covid text identification (CTI) is a crucial research concern in natural language processing (NLP). Social and electronic media are simultaneously adding a large volume of Covid-affiliated text on the World Wide Web due to the effortless access to the Internet, electronic gadgets and the Covid outbreak. Most of these texts are uninformative and contain misinformation, disinformation and malinformation that create an infodemic. Thus, Covid text identification is essential for controlling societal distrust and panic. Though very little Covid-related research (such as Covid disinformation, misinformation and fake news) has been reported in high-resource languages (e.g. English), CTI in low-resource languages (like Bengali) is in the preliminary stage to date. However, automatic CTI in Bengali text is challenging due to the deficit of benchmark corpora, complex linguistic constructs, immense verb inflexions and scarcity of NLP tools. On the other hand, the manual processing of Bengali Covid texts is arduous and costly due to their messy or unstructured forms. This research proposes a deep learning-based network (CovTiNet) to identify Covid text in Bengali. The CovTiNet incorporates an attention-based position embedding feature fusion for text-to-feature representation and attention-based CNN for Covid text identification. Experimental results show that the proposed CovTiNet achieved the highest accuracy of 96.61±.001% on the developed dataset (BCovC) compared to the other methods and baselines (i.e. BERT-M, IndicBERT, ELECTRA-Bengali, DistilBERT-M, BiLSTM, DCNN, CNN, LSTM, VDCNN and ACNN).

Shelterin reduces the accessibility of telomeric overhangs.

Telomeres terminate with a 50-300 bases long single-stranded G-rich overhang, which can be misrecognized as a DNA damage repair site. Shelterin plays critical roles in maintaining and protecting telomere ends by regulating access of various physiological agents to telomeric DNA, but the underlying mechanism is not well understood. Here, we measure how shelterin affects the accessibility of long telomeric overhangs by monitoring transient binding events of a short complementary peptide nucleic acid (PNA) probe using FRET-PAINT in vitro. We observed that the POT1 subunit of shelterin reduces the accessibility of the PNA probe by ∼2.5-fold, indicating that POT1 effectively binds to and protects otherwise exposed telomeric sequences. In comparison, a four-component shelterin stabilizes POT1 binding to the overhang by tethering POT1 to the double-stranded telomeric DNA and reduces the accessibility of telomeric overhangs by ∼5-fold. This enhanced protection suggests shelterin restructures the junction between single and double-stranded telomere, which is otherwise the most accessible part of the telomeric overhang.

Emerging accessibility patterns in long telomeric overhangs.

We present single-molecule experimental and computational modeling studies investigating the accessibility of human telomeric overhangs of physiologically relevant lengths. We studied 25 different overhangs that contain 4-28 repeats of GGGTTA (G-Tract) sequence and accommodate one to seven tandem G-quadruplex (GQ) structures. Using the FRET-PAINT method, we probed the distribution of accessible sites via a short imager strand, which is complementary to a G-Tract and transiently binds to available sites. We report accessibility patterns that periodically change with overhang length and interpret these patterns in terms of the underlying folding landscape and folding frustration. Overhangs that have [4n]G-Tracts, (12, 16, 20…) demonstrate the broadest accessibility patterns where the peptide nucleic acid probe accesses G-Tracts throughout the overhang. On the other hand, constructs with [4n+2]G-Tracts, (14, 18, 22…) have narrower patterns where the neighborhood of the junction between single- and double-stranded telomeres is most accessible. We interpret these results as the folding frustration being higher in [4n]G-Tract constructs compared to [4n+2]G-Tract constructs. We also developed a computational model that tests the consistency of different folding stabilities and cooperativities between neighboring GQs with the observed accessibility patterns. Our experimental and computational studies suggest the neighborhood of the junction between single- and double-stranded telomeres is least stable and most accessible, which is significant as this is a potential site where the connection between POT1/TPP1 (bound to single-stranded telomere) and other shelterin proteins (localized on double-stranded telomere) is established.

Consumers' Preferences for the Traceability Information of Seafood Safety.

Due to importing food and the perpetual changes from conventional wet markets to supermarkets in emerging markets, consumers have the opportunity to base their buying decisions on traceability systems. Seafood traceability systems involve information on production mode, inspection notes, sustainable sources, and sources of origin to provide consumer protection and help ensure that all seafood is safe to consume. This study aims to explore seafood markets by assessing the demand for traceability information attributes by utilising data from an experimental survey in an emerging market such as Bangladesh. The data were analysed using descriptive statistics, exploratory factor analysis, and a conditional logit model. The results demonstrate that consumers are concerned regarding vitamins, cholesterol, and preservatives, while they are little concerned about microbiological contamination, pesticide residues, genetic modification, and additives or artificial colours. The difference between the mean willingness to pay (WTP) for traditional and sustainable farmed fish is higher than that between the mean WTP for conventional and sustainable wild fish. In a ranked-choice voting system, the 'production mode' and 'claim of safety control (e.g., being formalin-free)' were the first and second most influential attributes in fish choices. The outcomes of the econometric model revealed that consumers are more likely to prefer traceability information about fish control (e.g., formalin-free), and they want to pay a price premium for this information. Alternatively, consumers are less likely to prefer farmed and imported fish, and their WTP for these fishes are highly inflated. This finding may be because consumers use wild and local origin as a cue for food safety or quality. This study hopes that the effects of such traceability information will optimise the production process and supply chain and help make seafood recall management more effective.

Decay of Piwi-Interacting RNAs in Human Cells Is Primarily Mediated by 5' to 3' Exoribonucleases.

Piwi-interacting RNAs (piRNAs) are a group of small noncoding RNA molecules that regulate the activity of transposons and control gene expression. The cellular concentration of RNAs is generally maintained by their rates of biogenesis and degradation. Although the biogenesis pathways of piRNAs have been well defined, their degradation mechanism is still unknown. Here, we show that degradation of human piRNAs is mostly dependent on the 5'-3' exoribonuclease pathway. The presence of 3'-end 2'-O-methylation in piRNAs significantly reduced their degradation through the exosome-mediated decay pathway. The accumulation of piRNAs in XRN1 and XRN2 exoribonuclease-depleted cells further supports the 5'-3' exoribonuclease-mediated decay of piRNAs. Moreover, formation of stable secondary structures in piRNAs slows the rate of XRN1-mediated degradation. Our findings establish a framework for the piRNA degradation mechanism in cells and thus provide crucial information about how the basal level concentration of piRNAs is maintained in cells.

Bone Marrow-Derived Cells Contribute to the Maintenance of Thymic Stroma including TECs.

In paradox to critical functions for T-cell selection and self-tolerance, the thymus undergoes profound age-associated atrophy and loss of T-cell function, further enhanced by cancer therapies. Identifying thymic epithelial progenitor populations capable of forming functional thymic tissue will be critical in understanding thymic epithelial cell (TEC) ontogeny and designing strategies to reverse involution. We identified a new population of progenitor cells, present in both the thymus and bone marrow (BM) of mice, that coexpress the hematopoietic marker CD45 and the definitive thymic epithelial marker EpCAM and maintain the capacity to form functional thymic tissue. Confocal analysis and qRT-PCR of sorted cells from both BM and thymus confirmed coexpression of CD45 and EpCAM. Grafting of C57BL/6 fetal thymi under the kidney capsule of H2BGFP transgenic mice revealed that peripheral CD45+ EpCAM+ GFP-expressing cells migrate into the developing thymus and contribute to both TECs and FSP1-expressing thymic stroma. Sorted BM-derived CD45+ EpCAM+ cells contribute to reaggregate thymic organ cultures (RTOCs) and differentiate into keratin and FoxN1-expressing TECs, demonstrating that BM cells can contribute to the maintenance of TEC microenvironments previously thought to be derived solely from endoderm. BM-derived CD45+ EpCAM+ cells represent a new source of progenitor cells that contribute to thymic homeostasis. Future studies will characterize the contribution of BM-derived CD45+ EpCAM+ TEC progenitors to distinct functional TEC microenvironments in both the steady-state thymus and under conditions of demand. Cell therapies utilizing this population may help counteract thymic involution in cancer patients.

Synthesis and Characterization of Cyclodextrin-Based Polyhemiaminal Composites with Enhanced Thermal Stability.

Polyhemiaminal (PHA) polymers are a new class of thermosetting polymers that have recently gained attention owing to their high mechanical strength and excellent recycling behavior. However, low thermal stability is a common issue in PHA polymers due to the thermally labile crosslinked knots. Herein, crosslinked PHA polymer composites were synthesized by reacting formaldehyde with a precursor solution of 4,4'-oxydianiline (ODA) and cyclodextrins (CDs) (α-, ß-, and γ-). The material obtained under optimal conditions (ODA:CD molar ratio of 1:0.5, 37% aqueous solution of formaldehyde (formalin)) exhibited good film formability and high thermal stability with two characteristic decomposition phenomena and a high char yield. The early decomposition of CDs and char formation led to high thermal stability. Time-resolved NMR analysis was conducted to study hemiaminal bond formation via a condensation reaction between ODA and formaldehyde. Furthermore, PHA matrix formation was confirmed by the dissolution of the deposited CD layer in a solution of N-methyl-2-pyrrolidinone containing 8-9 wt.% LiBr at 80 °C and FTIR analysis. Based on the elemental analysis results, PHA network formation was confirmed by considering a single unit of the PHA network with CD composition, including the solvent and water.

Essential Roles of Efferent Duct Multicilia in Male Fertility.

Cilia are microtubule-based hair-like organelles on the cell surface. Cilia have been implicated in various biological processes ranging from mechanosensation to fluid movement. Ciliary dysfunction leads to a plethora of human diseases, known as ciliopathies. Although non-motile primary cilia are ubiquitous, motile multicilia are found in restricted locations of the body, such as the respiratory tract, the oviduct, the efferent duct, and the brain ventricles. Multicilia beat in a whip-like motion to generate fluid flow over the apical surface of an epithelium. The concerted ciliary motion provides the driving force critical for clearing airway mucus and debris, transporting ova from the ovary to the uterus, maintaining sperm in suspension, and circulating cerebrospinal fluid in the brain. In the male reproductive tract, multiciliated cells (MCCs) were first described in the mid-1800s, but their importance in male fertility remained elusive until recently. MCCs exist in the efferent ducts, which are small, highly convoluted tubules that connect the testis to the epididymis and play an essential role in male fertility. In this review, we will introduce multiciliogenesis, discuss mouse models of male infertility with defective multicilia, and summarize our current knowledge on the biological function of multicilia in the male reproductive tract.

Reversal of G-Quadruplexes' Role in Translation Control When Present in the Context of an IRES.

G-quadruplexes (GQs) are secondary nucleic acid structures that play regulatory roles in various cellular processes. G-quadruplex-forming sequences present within the 5' UTR of mRNAs can function not only as repressors of translation but also as elements required for optimum function. Based upon previous reports, the majority of the 5' UTR GQ structures inhibit translation, presumably by blocking the ribosome scanning process that is essential for detection of the initiation codon. However, there are certain mRNAs containing GQs that have been identified as positive regulators of translation, as they are needed for translation initiation. While most cellular mRNAs utilize the 5' cap structure to undergo cap-dependent translation initiation, many rely on cap-independent translation under certain conditions in which the cap-dependent initiation mechanism is not viable or slowed down, for example, during development, under stress and in many diseases. Cap-independent translation mainly occurs via Internal Ribosomal Entry Sites (IRESs) that are located in the 5' UTR of mRNAs and are equipped with structural features that can recruit the ribosome or other factors to initiate translation without the need for a 5' cap. In this review, we will focus only on the role of RNA GQs present in the 5' UTR of mRNAs, where they play a critical role in translation initiation, and discuss the potential mechanism of this phenomenon, which is yet to be fully delineated.

BEmoC: A Corpus for Identifying Emotion in Bengali Texts.

Emotion classification in text has growing interest among NLP experts due to the enormous availability of people's emotions and its emergence on various Web 2.0 applications/services. Emotion classification in the Bengali texts is also gradually being considered as an important task for sports, e-commerce, entertainments, and security applications. However, It is a very critical task to develop an automatic emotion classification system for low-resource languages such as, Bengali. Scarcity of resources and deficiency of benchmark corpora make the task more complicated. Thus, the development of a benchmark corpus is the prerequisite to develop an emotion classifier for Bengali texts. This paper describes the development of an emotional corpus (hereafter called 'BEmoC') for classifying six emotions in Bengali texts. The corpus development process consists of four key steps: data crawling, pre-processing, labelling, and verification. A total of 7000 texts are labelled into six basic emotion categories such as anger, fear, surprise, sadness, joy, and disgust, respectively. Dataset evaluation with 0.969 Cohen's κ score indicates the close agreement between the corpus annotators and the expert. The analysis of evaluation also represents that the distribution of emotion words obeys Zipf's law. Moreover, the results of BEmoC analysis shown in terms of coding reliability, emotion density, and most frequent emotion words, respectively.

Spirulina ameliorates arsenic induced reproductive toxicity in male rats.

Spirulina (Spirulina platensis), has numerous health benefits including antioxidant, immunomodulatory, and anti-inflammatory activities, works against heavy metal toxicity, and is often used as a food supplement in human, animals, birds and fishes. This study aimed to evaluate the protective ability of the dietary spirulina against the toxic effects of inorganic arsenic (iAs) on male reproductive parameters in rats. Seventy-two mature Long-Evans male rats, dividing into six groups (T0, T1, T2, T3, T4 and T5) (12 rats/group) were included in this study. The T3, T4 and T5 group rats were treated with three consecutive doses (1.0 g, 1.5 g and 2.0 g/kg feed) of spirulina in feed along with 3.0 mg NaAsO2/kg body weight (BW) in drinking water (DW) daily for 90 days. Each rat of group T1 received NaAsO2 (3.0 mg/kg BW) in DW, and those of T2 group were fed with spirulina (2.0 g/kg feed) daily for 90 days. The rats of group T0 served as the control with normal feed and water. Total arsenic (tAs) contents, reproductive parameters (testicular weight, sperm motility and morphology), and histological changes in the testicles were evaluated in these rats. Arsenic dosing significantly (p=0.003, Kruskal-Wallis test) increased the tAs contents in the testicles, decreased testes weight, sperm morphology and motility compared to the controls. The effect of arsenic dosing was also evidenced by the histological changes like decreased germinal layers in the seminiferous tubules of the treated rats. Moreover, dietary spirulina (2.0 g/kg feed) supplementation significantly (p=0.011, Kruskal-Wallis test) lowered tAs contents in testicles and increases testes weights, sperm motility and morphology. Therefore, spirulina can be used as an effective dietary supplement to ameliorate the adverse effects of arsenic induced reproductive toxicities. However, further study is required to elucidate the underlying molecular mechanisms of reduction of arsenic induced reproductive toxicity by spirulina.

Loss of the ciliary protein Chibby1 in mice leads to exocrine pancreatic degeneration and pancreatitis.

Primary cilia protrude from the apical surface of many cell types and act as a sensory organelle that regulates diverse biological processes ranging from chemo- and mechanosensation to signaling. Ciliary dysfunction is associated with a wide array of genetic disorders, known as ciliopathies. Polycystic lesions are commonly found in the kidney, liver, and pancreas of ciliopathy patients and mouse models. However, the pathogenesis of the pancreatic phenotype remains poorly understood. Chibby1 (Cby1), a small conserved coiled-coil protein, localizes to the ciliary base and plays a crucial role in ciliogenesis. Here, we report that Cby1-knockout (KO) mice develop severe exocrine pancreatic atrophy with dilated ducts during early postnatal development. A significant reduction in the number and length of cilia was observed in Cby1-KO pancreta. In the adult Cby1-KO pancreas, inflammatory cell infiltration and fibrosis were noticeable. Intriguingly, Cby1-KO acinar cells showed an accumulation of zymogen granules (ZGs) with altered polarity. Moreover, isolated acini from Cby1-KO pancreas exhibited defective ZG secretion in vitro. Collectively, our results suggest that, upon loss of Cby1, concomitant with ciliary defects, acinar cells accumulate ZGs due to defective exocytosis, leading to cell death and progressive exocrine pancreatic degeneration after birth.

CEP164 is essential for efferent duct multiciliogenesis and male fertility.

Cilia are evolutionarily conserved microtubule-based structures that perform diverse biological functions. Cilia are assembled on basal bodies and anchored to the plasma membrane via distal appendages. In the male reproductive tract, multicilia in efferent ducts (EDs) move in a whip-like motion to prevent sperm agglutination. Previously, we demonstrated that the distal appendage protein CEP164 recruits Chibby1 (Cby1) to basal bodies to facilitate basal body docking and ciliogenesis. Mice lacking CEP164 in multiciliated cells (MCCs) (FoxJ1-Cre;CEP164fl/fl) show a significant loss of multicilia in the trachea, oviduct, and ependyma. In addition, we observed male sterility; however, the precise role of CEP164 in male fertility remained unknown. Here, we report that the seminiferous tubules and rete testis of FoxJ1-Cre;CEP164fl/fl mice exhibit substantial dilation, indicative of dysfunctional multicilia in the EDs. We found that multicilia were hardly detectable in the EDs of FoxJ1-Cre;CEP164fl/fl mice although FoxJ1-positive immature cells were present. Sperm aggregation and agglutination were commonly noticeable in the lumen of the seminiferous tubules and EDs of FoxJ1-Cre;CEP164fl/fl mice. In FoxJ1-Cre;CEP164fl/fl mice, the apical localization of Cby1 and the transition zone marker NPHP1 was severely diminished, suggesting basal body docking defects. TEM analysis of EDs further confirmed basal body accumulation in the cytoplasm of MCCs. Collectively, we conclude that male infertility in FoxJ1-Cre;CEP164fl/fl mice is caused by sperm agglutination and obstruction of EDs due to loss of multicilia. Our study, therefore, unravels an essential role of the distal appendage protein CEP164 in male fertility.

Encounters between Cas9/dCas9 and G-Quadruplexes: Implications for Transcription Regulation and Cas9-Mediated DNA Cleavage.

Using the nuclease-dead Cas9 (dCas9), we targeted in cellulo a G-rich sequence, which contains multiple potentially G-quadruplex (GQ) forming sites, within the human tyrosine hydroxylase (TH) promoter. We demonstrate that transcription can be up or down regulated by targeting different parts of this G-rich sequence. Our results suggest that TH transcription levels correlate with stability of different GQs formed by this sequence and targeting them with dCas9 can modulate their stability. Unlike alternative approaches, regulating TH expression by targeting the promoter GQs with dCas9 enables a specific and potentially transient control and does not require mutations in the sequence. We also investigated whether the presence of GQs in target sequences impacts DNA cleavage activity of Cas9. We discovered significant reduction in cleavage activity when the vicinity of a high-stability GQ was targeted. Furthermore, this reduction is significantly more prominent for the G-rich strand compared to the complementary C-rich strand.

Deep Phenotyping of Parkinson's Disease.

Phenotype is the set of observable traits of an organism or condition. While advances in genetics, imaging, and molecular biology have improved our understanding of the underlying biology of Parkinson's disease (PD), clinical phenotyping of PD still relies primarily on history and physical examination. These subjective, episodic, categorical assessments are valuable for diagnosis and care but have left gaps in our understanding of the PD phenotype. Sensors can provide objective, continuous, real-world data about the PD clinical phenotype, increase our knowledge of its pathology, enhance evaluation of therapies, and ultimately, improve patient care. In this paper, we explore the concept of deep phenotyping-the comprehensive assessment of a condition using multiple clinical, biological, genetic, imaging, and sensor-based tools-for PD. We discuss the rationale for, outline current approaches to, identify benefits and limitations of, and consider future directions for deep clinical phenotyping.

The PRR14 heterochromatin tether encodes modular domains that mediate and regulate nuclear lamina targeting.

A large fraction of epigenetically silent heterochromatin is anchored to the nuclear periphery via 'tethering proteins' that function to bridge heterochromatin and the nuclear membrane or nuclear lamina. We previously identified a human tethering protein, PRR14, that binds heterochromatin through an N-terminal domain, but the mechanism and regulation of nuclear lamina association remained to be investigated. Here we identify an evolutionarily conserved PRR14 nuclear lamina binding domain (LBD) that is both necessary and sufficient for positioning of PRR14 at the nuclear lamina. We show that PRR14 associates dynamically with the nuclear lamina, and provide evidence that such dynamics are regulated through phosphorylation and dephosphorylation of the LBD. Furthermore, we identify a PP2A phosphatase recognition motif within the evolutionarily conserved C-terminal Tantalus domain of PRR14. Disruption of this motif affects PRR14 localization to the nuclear lamina. The overall findings demonstrate a heterochromatin anchoring mechanism whereby the PRR14 tether simultaneously binds heterochromatin and the nuclear lamina through two separable modular domains. Our findings also describe an optimal PRR14 LBD fragment that could be used for efficient targeting of fusion proteins to the nuclear lamina.