The Molecular Architecture of the Nuclear Basket.

The nuclear pore complex (NPC) is the sole mediator of nucleocytoplasmic transport. Despite great advances in understanding its conserved core architecture, the peripheral regions can exhibit considerable variation within and between species. One such structure is the cage-like nuclear basket. Despite its crucial roles in mRNA surveillance and chromatin organization, an architectural understanding has remained elusive. Using in-cell cryo-electron tomography and subtomogram analysis, we explored the NPC's structural variations and the nuclear basket across fungi (yeast; S. cerevisiae), mammals (mouse; M. musculus), and protozoa (T. gondii). Using integrative structural modeling, we computed a model of the basket in yeast and mammals that revealed how a hub of Nups in the nuclear ring binds to basket-forming Mlp/Tpr proteins: the coiled-coil domains of Mlp/Tpr form the struts of the basket, while their unstructured termini constitute the basket distal densities, which potentially serve as a docking site for mRNA preprocessing before nucleocytoplasmic transport.

Identification and characterization of repurposed small molecule inhibitors of Mycobacterium tuberculosis caseinolytic protease B (ClpB) as anti-mycobacterials.

Mycobacterium tuberculosis (Mtb) caseinolytic protease B (ClpB) is a chaperone possessing a unique ability to resolubilize the aggregated proteins in vivo. ClpB has been shown to be important for the survival of Mtb within the host. Thus, it appears to be a promising target to develop new therapeutic molecules against tuberculosis. In this study, we have screened FDA approved compounds in silico to identify inhibitors against Mtb ClpB. In our screen, several compounds interacted with ClpB. The top four compounds, namely framycetin, gentamicin, ribostamycin and tobramycin showing the highest binding energy were selected for further investigation. MD simulations and tryptophan-based quenching of ClpB-drug complexes established that the selected inhibitors stably interacted with the target protein. The inhibitor and protein complexes were found to be stabilized by hydrogen bonding, and hydrophobic interactions. Although, the compounds did not affect the ATPase activity of ClpB significantly, the protein resolubilization activity of ClpB was remarkably reduced in their presence. All four compounds potently inhibited the growth of Mtb H37Ra. The antimycobacterial activity of the compounds appears to be due the inhibition of functional ClpB oligomer formation, in turn affecting its chaperonic activity.

Fermented formulation of Silybum marianum seeds: Optimization, heavy metal analysis, and hepatoprotective assessment.

BACKGROUND: Fermented formulations are extensively used in Ayurveda due to several benefits like improved palatability, bioavailability, pharmacological potential, and shelf life. These formulations can also quench the heavy metals from the plant material and thus reduce the toxicity. Seeds of Silybum marianum (L.) Gaertn. are widely used for the management of many liver diseases. STUDY DESIGN AND METHODS: In the present study, we developed a novel fermented formulation of S. marianum seeds and evaluated parameters like safety (heavy metal analysis) and effectiveness (hepatoprotective). As the developed formulation's validation is crucial, the critical process variables (time, pH, and sugar concentration) are optimized for alcohol and silybin content using the Box-Behnken design (BBD). RESULTS: The response surface methodology coupled with BBD predicted the optimized conditions (fermentation time (28 days), pH 5.6, and sugar concentration (22.04%)) for the development of a fermented formulation of the selected herb. Moreover, the alcohol content (6.5 ± 0.9%) and silybin concentration (26.1 ± 2.1%) were confirmed in optimized formulation by GC-MS and HPTLC analysis. The optimized formulation was also analyzed for heavy metals (Pb, As, Hg, and Cd); their concentration is significantly less than the decoction of herbs. Further, the comparative evaluation of the developed formulation with the marketed formulation also confirmed that the fermented formulation's silybin concentration and percentage release were significantly enhanced. In addition, the developed fermented formulation's percentage recovery of HepG2 cell lines after treatment with CCl4 was significantly improved compared with the marketed formulation. CONCLUSION: It can be summarized that the developed fermented formulation improves safety and effectiveness compared to other market formulations. Finally, it can be concluded that the developed fermented formulation could be further explored as a better alternative for developing Silybum marianum preparation.

Economic and life cycle cost analysis of building-integrated photovoltaic system for composite climatic conditions.

An insulated building-integrated photovoltaic (PV) roof prototype is designed, developed, and experimentally monitored for the composite climatic conditions in the current work. The prototype is monitored based on hourly indoor room temperature, relative humidity, discomfort index, decrement factor time lag, and power generation. To validate the results, a heat conduction equation was developed and simulated considering actual lower income group (LIG) building size and materials. Second-order polynomial equations were derived from simulation results to optimize insulation thickness. Additionally, the economic analysis of the insulated building-integrated Photovoltaic (BIPV) roof was analyzed and compared to the reinforced concrete cement (RCC) roof. The results reveal that insulated BIPV roofs outperform the RCC roof, reducing indoor temperatures by 3.34 ℃ to 1.37 ℃ within an optimum thickness range of 0.0838-0.1056 m. A time lag of 1 h and a significant reduction in decrement factor up to 0.29 are achieved. The average discomfort index of the proposed roof during sunshine hours was found to be between 23 and 26.5. The insulated BIPV roofs with levelized cost of electricity (LCOE) of the 3.38 Rs/kWh gave a payback period of 6.32 years and a higher internal rate of return of 29.4 compared to RCC roof. The current study increases the feasibility of PV modules to be used as building material.

Integrative spatiotemporal map of nucleocytoplasmic transport.

The Nuclear Pore Complex (NPC) facilitates rapid and selective nucleocytoplasmic transport of molecules as large as ribosomal subunits and viral capsids. It is not clear how key emergent properties of this transport arise from the system components and their interactions. To address this question, we constructed an integrative coarse-grained Brownian dynamics model of transport through a single NPC, followed by coupling it with a kinetic model of Ran-dependent transport in an entire cell. The microscopic model parameters were fitted to reflect experimental data and theoretical information regarding the transport, without making any assumptions about its emergent properties. The resulting reductionist model is validated by reproducing several features of transport not used for its construction, such as the morphology of the central transporter, rates of passive and facilitated diffusion as a function of size and valency, in situ radial distributions of pre-ribosomal subunits, and active transport rates for viral capsids. The model suggests that the NPC functions essentially as a virtual gate whose flexible phenylalanine-glycine (FG) repeat proteins raise an entropy barrier to diffusion through the pore. Importantly, this core functionality is greatly enhanced by several key design features, including 'fuzzy' and transient interactions, multivalency, redundancy in the copy number of FG nucleoporins, exponential coupling of transport kinetics and thermodynamics in accordance with the transition state theory, and coupling to the energy-reliant RanGTP concentration gradient. These design features result in the robust and resilient rate and selectivity of transport for a wide array of cargo ranging from a few kilodaltons to megadaltons in size. By dissecting these features, our model provides a quantitative starting point for rationally modulating the transport system and its artificial mimics.

Capsaicin binds the N-terminus of Hsp90, induces lysosomal degradation of Hsp70, and enhances the anti-tumor effects of 17-AAG (Tanespimycin).

Heat shock protein 90 (Hsp90) and its co-chaperones promote cancer, and targeting Hsp90 holds promise for cancer treatment. Most of the efforts to harness this potential have focused on targeting the Hsp90 N-terminus ATP binding site. Although newer-generation inhibitors have shown improved efficacy in aggressive cancers, induction of the cellular heat shock response (HSR) by these inhibitors is thought to limit their clinical efficacy. Therefore, Hsp90 inhibitors with novel mechanisms of action and that do not trigger the HSR would be advantageous. Here, we investigated the mechanism by which capsaicin inhibits Hsp90. Through mutagenesis, chemical modifications, and proteomic studies, we show that capsaicin binds to the N-terminus of Hsp90 and inhibits its ATPase activity. Consequently, capsaicin and its analogs inhibit Hsp90 ATPase-dependent progesterone receptor reconstitution in vitro. Capsaicin did not induce the HSR, instead, it promoted the degradation of Hsp70 through the lysosome-autophagy pathway. Remarkably, capsaicin did not induce degradation of the constitutively expressed cognate Hsc70, indicating selectivity for Hsp70. Combined treatments of capsaicin and the Hsp90 inhibitor 17-AAG improved the anti-tumor efficacy of 17-AAG in cell culture and tridimensional tumor spheroid growth assays using breast and prostate cancer models. Consistent with this, in silico docking studies revealed that capsaicin binding to the ATP binding site of Hsp90 was distinct from classical N-terminus Hsp90 inhibitors, indicating a novel mechanism of action. Collectively, these findings support the use of capsaicin as a chemical scaffold to develop novel Hsp90 N-terminus inhibitors as well as its ability to be a potential cancer co-therapeutic.

Comparison of the Microleakages of Four Root-End Filling Materials: An In Vitro Study.

INTRODUCTION: When a nonsurgical endodontic treatment is ineffective, surgery is necessary. This entails putting a retrofilling to seal the tooth's apex. Exposing the lesion, performing a curettage, exposing the root apex, resecting it, preparing the root end, and lastly filling the cavity with the proper material are all steps in endodontic surgery. Thus, the aim of this study is to compare the apical microleakage of four root-end filling materials in cavities prepared using ultrasonic retro tip in in vitro conditions. MATERIALS AND METHODS: An in vitro study was conducted on 60 extracted single-rooted teeth and was cut at the cementoenamel junction (CEJ). They were biomechanically prepared and obturated. Apical 3 mm root-end resection was done using a diamond disc. Root-end cavities were made using an ultrasonic retro tip. Teeth were separated into four groups and filled with SuperEBA®ï¸ ethoxy-benzoic acid (EBA; Keystone Industries, New Jersey), mineral trioxide aggregate (MTA), Biodentine (Septodont, France), and TotalFill Bioceramic Root Repair Material (BC RRM; FKG Dentaire Sàrl, Switzerland). The samples were kept in methylene blue dye and split longitudinally. The degree of dye penetration was observed under a stereomicroscope and scored. Finally, the results were analyzed. RESULTS: TotalFill BC RRM and Biodentine showed the least apical microleakage (p <0.05). Group 1 samples had the highest mean microleakage, followed by Group 2, Group 3, and Group 4 samples. CONCLUSION: All of the sample groups showed some evidence of microleakage, but not all of the samples showed leaking. SuperEBA (Group 1) demonstrated the highest microleakage when compared to the other groups.

Role of a substrate binding pocket in the amino terminal domain of Mycobacterium tuberculosis caseinolytic protease B (ClpB) in its function.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis when infects the host encounters several stresses within the host, resulting in aggregation of its proteins. To resolve this problem Mtb uses chaperones to either repair the damage or degrade the aggregated proteins. Mtb caseinolytic protein B (ClpB) helps in the prevention of aggregation and also resolubilization of aggregated proteins in bacteria, which is important for the survival of Mtb in the host. To function optimally, ClpB associates with its co-partners DnaK, DnaJ, and GrpE. The role of N-terminal domain (NTD) of Mtb ClpB in its function is not well understood. In this context, we investigated the interaction of three substrate mimicking peptides with the NTD of Mtb ClpB in silico. A substrate binding pocket, within the NTD of ClpB comprising of residues L136, R137, E138, K142, R144, R148, V149, Y158, and Y162 forming an ɑ-helix was thus identified. The residues L136 and R137 of the ɑ-helix were found to be important for the interaction of DnaK to ClpB. Further, nine single alanine recombinant variants of the identified residues were generated. As compared to the wild-type Mtb ClpB all the Mtb ClpB variants generated in this study were found to have reduced ATPase and protein refolding activity indicating the importance of the substrate binding pocket in ClpB function. The study demonstrates that the NTD of Mtb ClpB is important for its substrate interaction activity, and the substrate binding pocket identified in this study plays a crucial role in this interaction.Communicated by Ramaswamy H. Sarma.

ZEB1 potentiates chemoresistance in breast cancer stem cells by evading apoptosis.

Chemoresistance renders a challenge to the clinics to treat breast cancer patients. Current treatment strategies are effective in mitigating tumor growth but remain largely ineffective against cancer-initiating cells or breast Cancer Stem Cells (CSCs). Epithelial-to-mesenchymal-transition (EMT) regulates breast CSC physiology. Zinc finger E-box binding homeobox 1 (ZEB1) is a key EMT-transcription factor that regulates breast CSC - differentiation and metastasis. However, its potential role in modulating tumor chemoresistance has not yet been fully understood. In-silico analysis revealed a higher ZEB1 expression in breast cancer patients that leads to decreased overall and relapse-free survival. We generated sorted breast CSC with stable ZEB1 overexpression (CD24-/CD44+GFP-ZEB1) and/or silencing (CD24-/CD44+ZEB1 shRNA) as well as breast cancer cells with stable ZEB1 overexpression (CD24+GFP-ZEB1) and/or silencing (CD24+ZEB1 shRNA). An increased colony-forming efficiency and doxorubicin accumulation correlated with decreased promoter activity and expression profile of ABCC1 drug-efflux ABC transporter in CD24-/CD44+GFP-ZEB1. Additionally, CD24-/CD44+GFP-ZEB1 demonstrated doxorubicin-induced higher anti-apoptotic and lower pro-apoptotic protein expressions in the mitochondrial and cytosolic fractions. Chemoresistant CD24-/CD44+GFP-ZEB1 cells depicted 1000-fold higher IC-50 values of doxorubicin and decreased activation of JNK-p38 stress kinase molecular signaling-dependent mammosphere forming efficiency to evade apoptosis. Thus, ZEB1 and its downstream effectors are plausible therapeutic targets for the mitigation of breast cancer chemoresistance in patients.

Understanding the genomic selection for crop improvement: current progress and future prospects.

Although increased use of modern breeding techniques and technology has resulted in long-term genetic gain, the pace of genetic gain must be sped up to satisfy global agricultural demand. However, marker-assisted selection has proven its potential for improving qualitative traits with large effects regulated by one to few genes. Its contribution to the improvement of the quantitative traits regulated by a number of small-effect genes is modest. In this context, genomic selection (GS) has been regarded as the most promising method for genetically enhancing complicated features that are regulated by several genes, each of which has minor effects. By examining a population's phenotypes and high-density marker scores, genomic selection can forecast the breeding potential of individual lines. The fact that GS uses all marker data in the prediction model prevents skewed marker effect estimations and maximizes the amount of variation caused by small-effect QTL. It has the ability to speed up the breeding cycle and as a consequence of which superior genotypes are selected rapidly. Developing the best GS models while taking into account non-additive effects, genotype-by-environment interaction, and cost-effectiveness will enable the widespread implementation of GS in plants. These steps will also increase heritability estimation and prediction accuracy. This review focuses on the shift from conventional selection methods to GS, underlying statistical tools and methodologies, the state of GS research in agricultural plants, and prospects for its effective use in the creation of climate-resilient crops.

Productivity enhancement of solar still through heat transfer enhancement techniques in latent heat storage system: a review.

Solar still is one of the sustainable and renewable technology which converts brackish or salty water into fresh water. The technology helps in CO2 mitigation, global warming effect, and the use of solar desalination contributes towards decarbonization, mitigation of CO2 and other adverse global warming effect, and it contributes to the sustainable development goals (SDG). However, due to the low production rate of the distillate, the performance of solar still gets affected. The phase change materials (PCMs) as latent heat storage systems can enhance the thermal performance of solar still (SS). Further, techniques like increasing the area of contact and thermal conductivity can be practiced to enhance the heat transfer in PCM-SS. The article reviewed the performance of various designs of solar still integrated with PCM. Furthermore, the effect of nanoparticles enhanced PCM-integrated solar still with different absorber designs and configurations was seen. Compared to conventional solar still (CSS), the heat transfer techniques in PCM's SS can significantly improve the overall distillate productivity of Tubular SS by 218%, followed by single basin single slope SS 149%, pyramidal 125%, hemispherical 94%, and stepped 68%, respectively. In addition, the night time productivity was increased by 235%. Also, it was observed that in comparison to tubular PCM-SS, the nanodisbanded tubular PCM-SS increases the productivity by 68%, whereas in stepped solar still by using external condenser arrangement the productivity was increased by 48%. In single basin single slope, the nanoparticle disbanded PCMSS increases the productivity from 11 to 33%.

Surface water and groundwater interaction in the Kosi River alluvial fan of the Himalayan Foreland.

We report the isotopic composition of the surface water and groundwater of the Kosi River fan on the Himalayan Foreland, India. We have collected 65 water samples from surface water (Kosi River (n = 2), streams (n = 9), waterlogging (n = 29), and canal (n = 4)), and groundwater (n = 21) for δ18O and δ2H analysis during December 2019. We obtained groundwater level data measured at the observation wells from the Central Groundwater Board, India, for 1996 and 2017. The groundwater level varies from 1.0 to 8.1 m below ground level (bgl) and from 0.5 to 9.0 m bgl during 1996 and 2017, respectively. We have used water table fluctuation approach to estimate the recharge rate. The recharge rate in the Kosi Fan varies from 0.7 to 21.4 mm/year from 1996 to 2017. Further, we have used δ18O and δ2H values of water samples to identify the source and the interaction between surface water and groundwater. The δ18O value of groundwater shows a wide variation (from -9.3‰ to -5.6‰) compared to the surface water, i.e., streams (-7.8‰ to -6.4‰) and canals (-6.9‰ to -6.0‰), suggesting mixing in groundwater during recharge processes. Furthermore, we have used a two-component mixing model to assess the fraction contribution from streams and precipitation to groundwater. The estimated fraction contribution from stream water to groundwater ranges from 45 to 83%. We also suggest higher recharge is limited up to the depth of 6 m bgl. We suggest precipitation and surface water actively recharge groundwater. We conclude that marked spatial variation in the isotopic composition of groundwater is mainly due to the local recharge sources and interaction between surface water and groundwater.

Digital Eye Strain- A Comprehensive Review.

Digital eye strain (DES) is an entity encompassing visual and ocular symptoms arising due to the prolonged use of digital electronic devices. It is characterized by dry eyes, itching, foreign body sensation, watering, blurring of vision, and headache. Non-ocular symptoms associated with eye strain include stiff neck, general fatigue, headache, and backache. A variable prevalence ranging from 5 to 65% has been reported in the pre-COVID-19 era. With lockdown restrictions during the pandemic, outdoor activities were restricted for all age groups, and digital learning became the norm for almost 2 years. While the DES prevalence amongst children alone rose to 50-60%, the symptoms expanded to include recent onset esotropia and vergence abnormalities as part of the DES spectrum. New-onset myopia and increased progression of existing myopia became one of the most significant ocular health complications. Management options for DES include following correct ergonomics like reducing average daily screen time, frequent blinking, improving lighting, minimizing glare, taking regular breaks from the screen, changing focus to distance object intermittently, and following the 20-20-20 rule to reduce eye strain. Innovations in this field include high-resolution screens, inbuilt antireflective coating, matte-finished glass, edge-to-edge displays, and image smoothening graphic effects. Further explorations should focus on recommendations for digital screen optimization, novel spectacle lens technologies, and inbuilt filters to optimize visual comfort. A paradigm shift is required in our understanding of looking at DES from an etiological perspective, so that customized solutions can be explored accordingly. The aim of this review article is to understand the pathophysiology of varied manifestations, predisposing risk factors, varied management options, along with changing patterns of DES prevalence post COVID-19.

Immunological and safety profile of bacteriophage therapy: A pre-clinical study.

AIMS: Numerous pre-clinical and clinical studies have recently demonstrated the significant role of phage therapy in treating multidrug-resistant bacterial infections. However, only a few researchers have focused on monitoring the phage-mediated adverse reactions during phage therapy. The present study aimed to demonstrated the oral acute and sub-acute toxicity of bacteriophages (Klebsiella pneumoniae XDR strain) in Charles Foster rats with special reference to immunological response and adverse effects. METHODS AND RESULTS: Bacteriophages were orally administered in dosages of 1010  PFU/ml and a 1015  PFU/ml to Charles Foster rats as a single dose (in acute toxicity study) and daily dosage for 28 days (in sub-acute toxicity study). One millilitre suspension of bacteriophages was administered through the oral gavage feeding tube. No adverse effect was observed in any of the experimental as well as in the control animals. Furthermore, an insignificant change in food and water intake and body weight was observed throughout the study period compared with the control group rats. On the 28th day of phage administration, blood was collected to estimate haematological, biochemical and cytokines parameters. The data suggested no difference in the haematological, biochemical and cytokine profiles compared to the control group. No significant change in any of the treatment groups could be observed on the gross and histopathological examinations. The cytokines estimated, interleukin-1 beta (IL-1ß), IL-4, IL-6 and IFN-gamma, were found within the normal range during the experiment. CONCLUSIONS: The results concluded that no adverse effect, including the severe detrimental impact on oral administration of high (1010 PFU/ml) and very high dose (1015  PFU/ml) of the bacteriophages cocktail. SIGNIFICANCE AND IMPACT OF STUDY: The high and long-term oral administration of bacteriophages did not induce noticeable immunological response as well.

Video-based assessment of intraoperative surgical skill.

PURPOSE: Surgeons' skill in the operating room is a major determinant of patient outcomes. Assessment of surgeons' skill is necessary to improve patient outcomes and quality of care through surgical training and coaching. Methods for video-based assessment of surgical skill can provide objective and efficient tools for surgeons. Our work introduces a new method based on attention mechanisms and provides a comprehensive comparative analysis of state-of-the-art methods for video-based assessment of surgical skill in the operating room. METHODS: Using a dataset of 99 videos of capsulorhexis, a critical step in cataract surgery, we evaluated image feature-based methods and two deep learning methods to assess skill using RGB videos. In the first method, we predict instrument tips as keypoints and predict surgical skill using temporal convolutional neural networks. In the second method, we propose a frame-wise encoder (2D convolutional neural network) followed by a temporal model (recurrent neural network), both of which are augmented by visual attention mechanisms. We computed the area under the receiver operating characteristic curve (AUC), sensitivity, specificity, and predictive values through fivefold cross-validation. RESULTS: To classify a binary skill label (expert vs. novice), the range of AUC estimates was 0.49 (95% confidence interval; CI = 0.37 to 0.60) to 0.76 (95% CI = 0.66 to 0.85) for image feature-based methods. The sensitivity and specificity were consistently high for none of the methods. For the deep learning methods, the AUC was 0.79 (95% CI = 0.70 to 0.88) using keypoints alone, 0.78 (95% CI = 0.69 to 0.88) and 0.75 (95% CI = 0.65 to 0.85) with and without attention mechanisms, respectively. CONCLUSION: Deep learning methods are necessary for video-based assessment of surgical skill in the operating room. Attention mechanisms improved discrimination ability of the network. Our findings should be evaluated for external validity in other datasets.

Comprehensive structure and functional adaptations of the yeast nuclear pore complex.

Nuclear pore complexes (NPCs) mediate the nucleocytoplasmic transport of macromolecules. Here we provide a structure of the isolated yeast NPC in which the inner ring is resolved by cryo-EM at sub-nanometer resolution to show how flexible connectors tie together different structural and functional layers. These connectors may be targets for phosphorylation and regulated disassembly in cells with an open mitosis. Moreover, some nucleoporin pairs and transport factors have similar interaction motifs, which suggests an evolutionary and mechanistic link between assembly and transport. We provide evidence for three major NPC variants that may foreshadow functional specializations at the nuclear periphery. Cryo-electron tomography extended these studies, providing a model of the in situ NPC with a radially expanded inner ring. Our comprehensive model reveals features of the nuclear basket and central transporter, suggests a role for the lumenal Pom152 ring in restricting dilation, and highlights structural plasticity that may be required for transport.

Ethambutol toxicity: Expert panel consensus for the primary prevention, diagnosis and management of ethambutol-induced optic neuropathy.

Ethambutol use may lead to permanent vision loss by inducing a dose- and duration-dependent optic neuropathy. This has been of concern to ophthalmologists and physicians both; however, ethambutol continues to be used because of its anti-mycobacterial action with relative systemic safety. Recently, the guidelines of the Revised National Tuberculosis Control Programme of India have been revised to allow for fixed dose and longer duration of ethambutol use; this is likely to result in an increase in vision-threatening adverse effects. Taking cognizance of this, neuro-ophthalmologists, infectious disease specialists, and scientists met under the aegis of the Indian Neuro-Ophthalmology Society to deliberate on prevention, early diagnosis, and management of ethambutol-related toxic optic neuropathy. The recommendations made by the expert group focus on early suspicion of ethambutol toxicity through screening at the physician's office and opportunistic screening by the ophthalmologist. Further, they focus on an early diagnosis through identification of specific clinical biomarkers and on management in way of early stoppage of the drug and supportive therapy. This statement also describes the mechanism of reporting a case of toxic optic neuropathy through the Pharmacovigilance Programme of India and emphasizes the need for spreading awareness regarding vision-threatening adverse effects among patients and healthcare workers.

A viral genome packaging ring-ATPase is a flexibly coordinated pentamer.

Multi-subunit ring-ATPases carry out a myriad of biological functions, including genome packaging in viruses. Though the basic structures and functions of these motors have been well-established, the mechanisms of ATPase firing and motor coordination are poorly understood. Here, using single-molecule fluorescence, we determine that the active bacteriophage T4 DNA packaging motor consists of five subunits of gp17. By systematically doping motors with an ATPase-defective subunit and selecting single motors containing a precise number of active or inactive subunits, we find that the packaging motor can tolerate an inactive subunit. However, motors containing one or more inactive subunits exhibit fewer DNA engagements, a higher failure rate in encapsidation, reduced packaging velocity, and increased pausing. These findings suggest a DNA packaging model in which the motor, by re-adjusting its grip on DNA, can skip an inactive subunit and resume DNA translocation, suggesting that strict coordination amongst motor subunits of packaging motors is not crucial for function.

SNAI1-mediated transcriptional regulation of epithelial-to-mesenchymal transition genes in breast cancer stem cells.

BACKGROUND: Triple-negative breast cancer (TNBC) tumors are composed of a heterogeneous population containing both cancer cells and cancer stem cells (CSCs). These CSCs are generated through an epithelial-to-mesenchymal transition (EMT), thus making it pertinent to identify the unique EMT-molecular targets that regulate this phenomenon. METHODS AND RESULTS: In the present study, we performed in silico analysis of microarray data from luminal, Her2+, and TNBC cell lines and identified 15 relatively unexplored EMT-related differentially expressed genes (DEGs) along with the markedly high expression of EMT-transcription factor (EMT-TF), SNAI1. Interestingly, stable overexpression of SNAI1 in MCF-7 induced the expression of DEGs along with increased migration, invasion, and in vitro tumorigenesis that was comparable to TNBCs. Next, stable SNAI1 overexpression led to increased expression of DEGs that was reverted with SNAI1 silencing in both breast cancer cells and CSCs sorted from various TNBC cell lines. Higher fold enrichment of SNAI1 on E-boxes in the promoter regions suggested a positive regulation of ALCAM, MMP2, MMP13, MMP14, VCAN, ANKRD1, KRT16, CTGF, TGFRIIß, PROCR negative regulation of CDH1, DSP and DSC3B by SNAI1 leading to EMT. Furthermore, SNAI1-mediated increased migration, invasion, and tumorigenesis in these sorted cells led to the activation of signaling mediators, ERK1/2, STAT3, Src, and FAK. Finally, the SNAI1-mediated activation of breast CSC phenotypes was perturbed by inhibition of downstream target, MMPs using Ilomastat. CONCLUSION: Thus, the molecular investigation for the gene regulatory framework in the present study identified MMPs, a downstream effector in the SNAI1-mediated EMT regulation.