A new lineage nomenclature to aid genomic surveillance of dengue virus.

Dengue virus (DENV) is currently causing epidemics of unprecedented scope in endemic settings and expanding to new geographical areas. It is therefore critical to track this virus using genomic surveillance. However, the complex patterns of viral genomic diversity make it challenging to use the existing genotype classification system. Here, we propose adding 2 sub-genotypic levels of virus classification, named major and minor lineages. These lineages have high thresholds for phylogenetic distance and clade size, rendering them stable between phylogenetic studies. We present assignment tools to show that the proposed lineages are useful for regional, national, and subnational discussions of relevant DENV diversity. Moreover, the proposed lineages are robust to classification using partial genome sequences. We provide a standardized neutral descriptor of DENV diversity with which we can identify and track lineages of potential epidemiological and/or clinical importance. Information about our lineage system, including methods to assign lineages to sequence data and propose new lineages, can be found at: dengue-lineages.org.

Role of specific CDKs in regulating DNA damage repair responses and replication stress.

Cyclins along with their catalytic units, Cyclin-dependent kinases (CDKs) regulate the cell cycle transition and transcription; and are essentially known as 'master regulators' in modulating DNA damage response (DDR) and replication stress. In addition to influencing DNA repair and damage signaling, CDKs also play a pivotal role in cell division fidelity and the maintenance of genomic integrity after DNA damage. In this review, we focus on the intricate ways by which specific CDKs mainly CDK7, CDK9, and CDK12/13, regulate the cell cycle progression and transcription and how their modulation can lead to lethal effects on the integrity of the genome. With a better knowledge of how these CDKs control the DDR and replication stress, it is now possible to combine CDK inhibitors with chemotherapeutic drugs that damage DNA in ways that can be applied in clinical settings as successful therapeutic strategies.

Elastic contribution of polymeric fluids augments salinity-gradient-induced electric potential across a microfluidic channel.

HYPOTHESIS: Harnessing electrical energy from salinity gradients, particularly for powering micro and nanoscale devices, has become a focal point of recent research attention, due to its renewable and biocompatible nature. Much of the reported research in that direction revolves around optimizing the membrane architecture and the charge distribution to maximize the induced electric potential, with no particular emphasis on the fluid rheology. However, many of the modern miniature systems, typically the bio-inspired ones, concern fluids with complex rheological characteristics, where the results for Newtonian solvents may not trivially apply. Here, we hypothesize that the interplay between interfacial electro-mechanics and the fluid rheology can influence the effectiveness of salinity-gradient-modulated electrokinetics significantly - an aspect that has largely remained overlooked. THEORY AND EXPERIMENTS: Here we report the first experiments supplemented by a theoretical model that unveil how that the addition of polymers in a solvent modulates the salinity gradient - induced electric potential in a microfluidic channel. Our theoretical framework considers the simplified Phan-Thien Tanner (sPTT) constitutive model, which represents the viscoelastic characteristics of fluids. Experiments were conducted with combined pressure driven and salinity gradient driven flow through microchannel involving dilute solutions of polyethylene oxide (PEO) of different molecular weights and concentrations to successfully validate the theoretical approach. FINDINGS: Our findings indicate that the induced electrical potential increased non-linearly with the saline concentration ratio across the microchannel, as compared traditional linear response. Our results demonstrate how the elasticity of fluid may enable realizing an optimal benefit to this effect, by arresting the viscous resistance and uplifting the elastic response via utilizing polymeric inclusions of high relaxation times. These results provide specific insights on preferential windows of augmenting the induced streaming potential by harnessing the viscoelastic nature of the solution and the imposed salt concentration, bearing critical implications in miniature energy harvesting and desalination technology.

A new lineage nomenclature to aid genomic surveillance of dengue virus.

Dengue virus (DENV) is currently causing epidemics of unprecedented scope in endemic settings and expanding to new geographical areas. It is therefore critical to track this virus using genomic surveillance. However, the complex patterns of viral genomic diversity make it challenging to use the existing genotype classification system. Here we propose adding two sub-genotypic levels of virus classification, named major and minor lineages. These lineages have high thresholds for phylogenetic distance and clade size, rendering them stable between phylogenetic studies. We present an assignment tool to show that the proposed lineages are useful for regional, national and sub-national discussions of relevant DENV diversity. Moreover, the proposed lineages are robust to classification using partial genome sequences. We provide a standardized neutral descriptor of DENV diversity with which we can identify and track lineages of potential epidemiological and/or clinical importance. Information about our lineage system, including methods to assign lineages to sequence data and propose new lineages, can be found at: dengue-lineages.org.

Light-induced electronic polarization in antiferromagnetic Cr2O3.

In a solid, the electronic subsystem can exhibit incipient order with lower point group symmetry than the crystal lattice. Ultrafast external fields that couple exclusively to electronic order parameters have rarely been investigated, however, despite their potential importance in inducing exotic effects. Here we show that when inversion symmetry is broken by the antiferromagnetic order in Cr2O3, transmitting a linearly polarized light pulse through the crystal gives rise to an in-plane rotational symmetry-breaking (from C3 to C1) via optical rectification. Using interferometric time-resolved second harmonic generation, we show that the ultrafast timescale of the symmetry reduction is indicative of a purely electronic response; the underlying spin and crystal structures remain unaffected. The symmetry-broken state exhibits a dipole moment, and its polar axis can be controlled with the incident light. Our results establish a coherent nonlinear optical protocol by which to break electronic symmetries and produce unconventional electronic effects in solids.

Transcription elongation defects link oncogenic SF3B1 mutations to targetable alterations in chromatin landscape.

Transcription and splicing of pre-messenger RNA are closely coordinated, but how this functional coupling is disrupted in human diseases remains unexplored. Using isogenic cell lines, patient samples, and a mutant mouse model, we investigated how cancer-associated mutations in SF3B1 alter transcription. We found that these mutations reduce the elongation rate of RNA polymerase II (RNAPII) along gene bodies and its density at promoters. The elongation defect results from disrupted pre-spliceosome assembly due to impaired protein-protein interactions of mutant SF3B1. The decreased promoter-proximal RNAPII density reduces both chromatin accessibility and H3K4me3 marks at promoters. Through an unbiased screen, we identified epigenetic factors in the Sin3/HDAC/H3K4me pathway, which, when modulated, reverse both transcription and chromatin changes. Our findings reveal how splicing factor mutant states behave functionally as epigenetic disorders through impaired transcription-related changes to the chromatin landscape. We also present a rationale for targeting the Sin3/HDAC complex as a therapeutic strategy.

Rapid detection of Mucorales in human blood and urine samples by functionalized Heusler magnetic nanoparticle assisted customized loop-mediated isothermal amplification.

Mucormycosis is a rare disease with scarce diagnostic methods for early intervention. Available strategies employing direct microscopy using calcofluor white-KOH, culture, radiologic, and histopathologic testing often are time-intensive and demand intricate protocols. Nucleic Acid Amplification Test holds promise due to its high sensitivity combined with rapid detection. Loop-mediated isothermal amplification (LAMP) based detection offers an ultrasensitive technique that does not require complicated thermocyclers like in polymerase chain reaction, offering a straightforward means for improving diagnoses as a near-point-of-care test. The study introduces a novel magnetic nanoparticle-based LAMP assay for carryover contaminant capture to reduce false positives. Solving the main drawback of LAMP-based diagnosis techniques. The assay targets the cotH gene, which is invariably specific to Mucorales. The assay was tested with various species of Mucorales, and the limit of detections for Rhizopus microsporus, Lichtheimia corymbifera, Rhizopus arrhizus, Rhizopus homothallicus, and Cunninghamella bertholletiae were 1 fg, 1 fg, 0.1 pg, 0.1 pg, and 0.01 ng, respectively. This was followed by a clinical blindfolded study using whole blood and urine samples from 30 patients diagnosed with Mucormycosis. The assay has a high degree of repeatability and had an overall sensitivity of > 83%. Early Mucormycosis detection is crucial, as current lab tests from blood and urine lack sensitivity and take days for confirmation despite rapid progression and severe complications. Our developed technique enables the confirmation of Mucormycosis infection in < 45 min, focusing specifically on the RT-LAMP process. Consequently, this research offers a viable technique for quickly identifying Mucormycosis from isolated DNA of blood and urine samples instead of invasive tissue samples.

Mucormycosis is a challenging disease to diagnose early. This study introduces a sensitive and rapid diagnostic approach using Loop-mediated isothermal amplification technology. Testing blood and urine samples from 30 patients revealed promising sensitivity and repeatability, indicating its potential for non-invasive diagnosis.

Sperm intrusion into the implantation-stage blastocyst and its potential biological significance.

The human embryo derives from fusion of oocyte and sperm, undergoes growth and differentiation, resulting in a blastocyst. To initiate implantation, the blastocyst hatches from the zona pellucida, allowing access from external inputs. Modelling of uterine sperm distribution indicates that 200-5000 sperm cells may reach the implantation-stage blastocyst following natural coitus. We show ultrastructural evidence of sperm cells intruding into trophectoderm cells of zona-free blastocysts obtained from the uterus of rhesus monkeys. Interaction between additional sperm and zona-free blastocyst could be an evolutionary feature yielding adaptive processes influencing the developmental fate of embryos. This process bears potential implications in pregnancy success, sperm competition and human health.

131I dose coefficients for a reference population using age-specific models.

Age-specific dose coefficients are required to assess internal exposure to the general public. This study utilizes reference age-specific biokinetic models of iodine to estimate the total number of nuclear disintegrations ã(rS,τ) occurring in source regions (rS) during the commitment time (τ). Age-specific S values are estimated for 35 target regions due to131I present in 22rSusing data from 10 paediatric reference computational phantoms (representing five ages for both sexes) published recently by the International Commission of Radiation Protection (ICRP). Monte Carlo transport simulations are performed in FLUKA code. The estimated ã(rS,τ) and S values are then used to compute the committed tissue equivalent dose HT(τ) for 27 radiosensitive tissues and dose coefficients e(τ) for all five ages due to inhalation and ingestion of131I. The derived ã(rS,τ) values in the thyroid source are observed to increase with age due to the increased retention of iodine in the thyroid. S values are found to decrease with age, mainly due to an increase in target masses. Generally, HT(τ) values are observed to decrease with age, indicating the predominant behaviour of S values over ã(rS,τ). On average, ingestion dose coefficients are 63% higher than for inhalation in all ages. The maximum contribution to dose coefficients is from the thyroid, accounting for 96% in the case of newborns and 98%-99% for all other ages. Furthermore, the estimated e(τ) values for the reference population are observed to be lower than previously published reference values from the ICRP. The estimated S, HT(τ) and e(τ) values can be used to improve estimations of internal doses to organs/whole body for members of the public in cases of131I exposure. The estimated dose coefficients can also be interpolated for other ages to accurately evaluate the doses received by the general public during131I therapy or during a radiological emergency.

3D Cyclophane for the Selective Conversion of Epoxide to Cyclic Carbonate.

A novel three-dimensional (3D) cyclophane molecule 1 was synthesized and fully characterized. Cyclophane 1, which can form a N heterocyclic carbene, was tested for conversion of certain epoxides (3-6) [scheme 2] to cyclic carbonates in the presence of CO2. Propylene oxide (3) was found to have more reactivity with cyclophane 1 compared to the other epoxides. The theoretical calculations based on N,N'-disubstituted imidazol(in)ium-2-carboxylates derived from N,N' disubstituted imidazole as the source of N-heterocyclic carbene show lower activation energy in the case of the reactivity of epoxides 5 and 6 as compared to 3 and 4. However, cyclophane 1, which possesses a 3D geometry, can form the open intermediate with CO2 and propylene oxide more feasibly than the other three epoxides, which have larger sizes as compared to propylene oxide. Hence, the reaction of propylene oxide, CO2, and cyclophane 1 can follow the mechanistic path 1, whereas the epoxides 4-6 can follow a different mechanistic path 2. Cyclophane 1 is the first example of a cyclophane to act as an organocatalyst for the conversion of CO2 and epoxide to cyclic carbonate via the N heterocyclic carbene pathway.

Cholesterol catalyzes unfolding in membrane-inserted motifs of the pore forming protein cytolysin A.

Plasma membrane-induced protein folding and conformational transitions play a central role in cellular homeostasis. Several transmembrane proteins are folded in the complex lipid milieu to acquire a specific structure and function. Bacterial pore forming toxins (PFTs) are proteins expressed by a large class of pathogenic bacteria that exploit the plasma membrane environment to efficiently undergo secondary structure changes, oligomerize, and form transmembrane pores. Unregulated pore formation causes ion imbalance, leading to cell death and infection. Determining the free energy landscape of these membrane-driven-driven transitions remains a challenging problem. Although cholesterol recognition is required for lytic activity of several proteins in the PFT family of toxins, the regulatory role of cholesterol for the α-PFT, cytolysin A expressed by Escherichia coli remains unexplained. In a recent free energy computation, we showed that the ß tongue, a critical membrane-inserted motif of the ClyA toxin, has an on-pathway partially unfolded intermediate that refolds into the helix-turn-helix motif of the pore state. To understand the molecular role played by cholesterol, we carry out string-method-based computations in membranes devoid of cholesterol, which reveals an increase of ∼30 times in the free energy barrier for the loss of ß sheet secondary structure when compared with membranes containing cholesterol. Specifically, the tyrosine-cholesterol interaction was found to be critical to creating the unfolded intermediate. Cholesterol also increases the packing and hydrophobicity of the bilayer, resulting in enhanced interactions of the bound protein before complete membrane insertion. Our study illustrates that cholesterol is critical to catalyzing and stabilizing the membrane-inserted unfolded state of the ß tongue motif of ClyA, opening up fresh insights into cholesterol-assisted unfolding of membrane proteins.

Droplet-on-chip electro-spectroscopy detects the ultra-short relaxation time of a dilute polymer solution.

We report an electrode-embedded on-chip platform technology for the precise determination of ultra-short (of the order of a few nanoseconds) relaxation times of dilute polymer solutions, by deploying time-alternating electrical voltages. Our methodology delves into the sensitive dependence of the contact line dynamics of a droplet of the polymer solution atop a hydrophobic interface in response to the actuation voltage, resulting in a non-trivial interplay between the time-evolving electrical, capillary, and viscous forces. This culminates into a time-decaying dynamic response that mimics the features of a damped oscillator having its 'stiffness' mapped with the polymeric content of the droplet. The observed electro-spreading characteristics of the droplet are thus shown to correlate explicitly with the relaxation time of the polymer solution, drawing analogies with a damped electro-mechanical oscillator. By corroborating well with the reported values of the relaxation times as obtained from more elaborate and sophisticated laboratory set-ups. Our findings provide perspectives for a unique and simple approach towards electrically-modulated on-chip-spectroscopy for deriving ultra-short relaxation times of a broad class of viscoelastic fluids that could not be realized thus far.

Evidence for Bootstrap Percolation Dynamics in a Photoinduced Phase Transition.

Upon intense femtosecond photoexcitation, a many-body system can undergo a phase transition through a nonequilibrium route, but understanding these pathways remains an outstanding challenge. Here, we use time-resolved second harmonic generation to investigate a photoinduced phase transition in Ca_{3}Ru_{2}O_{7} and show that mesoscale inhomogeneity profoundly influences the transition dynamics. We observe a marked slowing down of the characteristic time τ that quantifies the transition between two structures. τ evolves nonmonotonically as a function of photoexcitation fluence, rising from below 200 fs to ∼1.4 ps, then falling again to below 200 fs. To account for the observed behavior, we perform a bootstrap percolation simulation that demonstrates how local structural interactions govern the transition kinetics. Our work highlights the importance of percolating mesoscale inhomogeneity in the dynamics of photoinduced phase transitions and provides a model that may be useful for understanding such transitions more broadly.

Evolutionary dynamics of dengue virus in India.

More than a hundred thousand dengue cases are diagnosed in India annually, and about half of the country's population carries dengue virus-specific antibodies. Dengue propagates and adapts to the selection pressures imposed by a multitude of factors that can lead to the emergence of new variants. Yet, there has been no systematic analysis of the evolution of the dengue virus in the country. Here, we present a comprehensive analysis of all DENV gene sequences collected between 1956 and 2018 from India. We examine the spatio-temporal dynamics of India-specific genotypes, their evolutionary relationship with global and local dengue virus strains, interserotype dynamics and their divergence from the vaccine strains. Our analysis highlights the co-circulation of all DENV serotypes in India with cyclical outbreaks every 3-4 years. Since 2000, genotype III of DENV-1, cosmopolitan genotype of DENV-2, genotype III of DENV-3 and genotype I of DENV-4 have been dominating across the country. Substitution rates are comparable across the serotypes, suggesting a lack of serotype-specific evolutionary divergence. Yet, the envelope (E) protein displays strong signatures of evolution under immune selection. Apart from drifting away from its ancestors and other contemporary serotypes in general, we find evidence for recurring interserotype drift towards each other, suggesting selection via cross-reactive antibody-dependent enhancement. We identify the emergence of the highly divergent DENV-4-Id lineage in South India, which has acquired half of all E gene mutations in the antigenic sites. Moreover, the DENV-4-Id is drifting towards DENV-1 and DENV-3 clades, suggesting the role of cross-reactive antibodies in its evolution. Due to the regional restriction of the Indian genotypes and immunity-driven virus evolution in the country, ~50% of all E gene differences with the current vaccines are focused on the antigenic sites. Our study shows how the dengue virus evolution in India is being shaped in complex ways.

Post-secretory synthesis of a natural analog of iron-gall ink in the black nectar of Melianthus spp.

The black nectar produced by Melianthus flowers is thought to serve as a visual attractant to bird pollinators, but the chemical identity and synthesis of the black pigment are unknown. A combination of analytical biochemistry, transcriptomics, proteomics, and enzyme assays was used to identify the pigment that gives Melianthus nectar its black color and how it is synthesized. Visual modeling of pollinators was also used to infer a potential function of the black coloration. High concentrations of ellagic acid and iron give the nectar its dark black color, which can be recapitulated through synthetic solutions containing only ellagic acid and iron(iii). The nectar also contains a peroxidase that oxidizes gallic acid to form ellagic acid. In vitro reactions containing the nectar peroxidase, gallic acid, hydrogen peroxide, and iron(iii) fully recreate the black color of the nectar. Visual modeling indicates that the black color is highly conspicuous to avian pollinators within the context of the flower. Melianthus nectar contains a natural analog of iron-gall ink, which humans have used since at least medieval times. This pigment is derived from an ellagic acid-Fe complex synthesized in the nectar and is likely involved in the attraction of passerine pollinators endemic to southern Africa.

Transcription elongation defects link oncogenic splicing factor mutations to targetable alterations in chromatin landscape.

Transcription and splicing of pre-messenger RNA are closely coordinated, but how this functional coupling is disrupted in human disease remains unexplored. Here, we investigated the impact of non-synonymous mutations in SF3B1 and U2AF1, two commonly mutated splicing factors in cancer, on transcription. We find that the mutations impair RNA Polymerase II (RNAPII) transcription elongation along gene bodies leading to transcription-replication conflicts, replication stress and altered chromatin organization. This elongation defect is linked to disrupted pre-spliceosome assembly due to impaired association of HTATSF1 with mutant SF3B1. Through an unbiased screen, we identified epigenetic factors in the Sin3/HDAC complex, which, when modulated, normalize transcription defects and their downstream effects. Our findings shed light on the mechanisms by which oncogenic mutant spliceosomes impact chromatin organization through their effects on RNAPII transcription elongation and present a rationale for targeting the Sin3/HDAC complex as a potential therapeutic strategy. HIGHLIGHTS: Oncogenic mutations of SF3B1 and U2AF1 cause a gene-body RNAPII elongation defectRNAPII transcription elongation defect leads to transcription replication conflicts, DNA damage response, and changes to chromatin organization and H3K4me3 marksThe transcription elongation defect is linked to disruption of the early spliceosome formation through impaired interaction of HTATSF1 with mutant SF3B1.Changes to chromatin organization reveal potential therapeutic strategies by targeting the Sin3/HDAC pathway.

VPS45 is required for both diffuse and tip growth of Arabidopsis thaliana cells.

Introduction: VPS45 belongs to the Sec1/Munc18 family of proteins, which interact with and regulate Qa-SNARE function during membrane fusion. We have shown previously that Arabidopsis thaliana VPS45 interacts with the SYP61/SYP41/VTI12 SNARE complex, which locates on the trans-Golgi network (TGN). It is required for SYP41 stability, and it functions in cargo trafficking to the vacuole and in cell expansion. It is also required for correct auxin distribution during gravitropism and lateral root growth. Results: As vps45 knockout mutation is lethal in Arabidopsis, we identified a mutant, vps45-3, with a point mutation in the VPS45 gene causing a serine 284-to-phenylalanine substitution. The VPS45-3 protein is stable and maintains interaction with SYP61 and SYP41. However, vps45-3 plants display severe growth defects with significantly reduced organ and cell size, similar to vps45 RNAi transgenic lines that have reduced VPS45 protein levels. Root hair and pollen tube elongation, both processes of tip growth, are highly compromised in vps45-3. Mutant root hairs are shorter and thicker than those of wild-type plants, and are wavy. These root hairs have vacuolar defects, containing many small vacuoles, compared with WT root hairs with a single large vacuole occupying much of the cell volume. Pollen tubes were also significantly shorter in vps45-3 compared to WT. Discussion: We thus show that VPS45 is essential for proper tip growth and propose that the observed vacuolar defects lead to loss of the turgor pressure needed for tip growth.

Conformational Flexibility Is a Key Determinant for the Lytic Activity of the Pore-Forming Protein, Cytolysin A.

Several bacterial infections are mediated by pore-forming toxins (PFTs), a subclass of proteins that oligomerize on mammalian cell membranes forming lytic nanopores. Cytolysin A (ClyA), an α-PFT, undergoes a dramatic conformational change restructuring its two membrane-binding motifs (the ß-tongue and the N-terminus helix), during pore formation. A complete molecular picture for this key transition and the driving force behind the secondary structure change upon membrane binding remain elusive. Using all-atom molecular dynamics (MD) simulations of the ClyA monomer and string method based free energy computations with path collective variables, we illustrate that an unfolded ß-tongue motif is an on-pathway intermediate during the transition to the helix-turn-helix motif of the protomer. An aggregate of 28 µs of all-atom thermal unfolding MD simulations of wild-type ClyA and its single point mutants reveal that the membrane-binding motifs of the ClyA protein display high structural flexibility in water. However, point mutations in these motifs lead to a distinct reduction in the flexibility, especially in the ß-tongue, thereby stabilizing the pretransition secondary structure. Resistance to unfolding was further corroborated by MD simulations of the ß-tongue mutant motif in the membrane. Combined with the thermal unfolding simulations, we posit that the ß-tongue as well as N-terminal mutants that lower the tendency to unfold and disorder the ß-tongue are detrimental to pore formation by ClyA and its lytic activity. Erythrocyte turbidity and vesicle leakage assays indeed reveal a loss of activity for the ß-tongue mutant, and delayed kinetics for the N-terminus mutants. On the other hand, a point mutation in the extracellular domain that did not abrogate lytic activity displayed similar unfolding characteristics as the wild type. Thus, attenuation of conformational flexibility in membrane-binding motifs correlates with reduced lytic and leakage activity. Combined with secondary structure changes observed in the membrane bound states, our study shows that the tendency to unfold in the ß-tongue region is a critical step in the conformational transition and bistability of the ClyA protein and mutants that disrupt this tendency reduced pore formation. Overall, our finding suggests that inherent flexibility in the protein could play a wider and hitherto unrecognized role in membrane-mediated conformational transitions of PFTs and other membrane protein transformations.

A Versatile Suspended Lipid Membrane System for Probing Membrane Remodeling and Disruption.

Artificial membrane systems can serve as models to investigate molecular mechanisms of different cellular processes, including transport, pore formation, and viral fusion. However, the current, such as SUVs, GUVs, and the supported lipid bilayers suffer from issues, namely high curvature, heterogeneity, and surface artefacts, respectively. Freestanding membranes provide a facile solution to these issues, but current systems developed by various groups use silicon or aluminum oxide wafers for fabrication that involves access to a dedicated nanolithography facility and high cost while conferring poor membrane stability. Here, we report the development, characterization and applications of an easy-to-fabricate suspended lipid bilayer (SULB) membrane platform leveraging commercial track-etched porous filters (PCTE) with defined microwell size. Our SULB system offers a platform to study the lipid composition-dependent structural and functional properties of membranes with exceptional stability. With dye entrapped in PCTE microwells by SULB, we show that sphingomyelin significantly augments the activity of pore-forming toxin, Cytolysin A (ClyA) and the pore formation induces lipid exchange between the bilayer leaflets. Further, we demonstrate high efficiency and rapid kinetics of membrane fusion by dengue virus in our SULB platform. Our suspended bilayer membrane mimetic offers a novel platform to investigate a large class of biomembrane interactions and processes.