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Nag, S.

Publications and source records attributed to Nag, S..

At least 19 recordsLinked to original sources

Variational Autoencoder-enabled High-throughput Drug Screening for HIV Latency Modulators predicted through Noise in Gene Expression

Due to its ability to establish a pool of undetectable and latently infected cells that can initiate viral production through random reactivation, a cure to human immunodeficiency virus (HIV) infections has remained elusive. Many approaches have been proposed, including the "shock and kill" method where latency reversing agents (LRAs) are administered to reactivate latently infected cells out of latency and remove them through immune targeting and clearance, and the "block and lock" method where latency promoting agents (LPAs) are administered to inhibit reactivation and potentially induce a "deep latency" state where infected cells can no longer reactivate. Previous large scale drug screen studies have demonstrated a correlation between a compounds capability to modulate the fluctuations (or "noise") in HIV gene expression and its potential to modulate HIV latency. However, measurements of gene expression noise are labor-and cost-intensive. To circumvent these drawbacks, we trained a variational autoencoder (VAE) on a previously published large scale time-lapse fluorescence microscopy dataset, and performed an in silico screening of [~]175,000 compounds for HIV latency modulators. Out of the top 113 predicted modulators that were experimentally tested, 16 latency reversing agent (LRA) synergizers and 2 latency promoting agents (LPAs) were confirmed, yielding an overall experimental hit rate of 15.9%. Our work demonstrates that in silico drug screening modalities, guided by existing large-scale experimental datasets, can yield high experimental hit rates, reducing costs incurred from labor-intensive wet lab-focused methodologies.

biochemistry↗

Porcine Left Atrial and Ventricular Thick Filaments Exhibit Distinct Resting Structures and Calcium-dependent Responses

The heart maintains systemic perfusion through the coordinated function of its four chambers: the left and right atria and ventricles. Each chamber has distinct structural, functional, and molecular properties tailored to its role in circulation, which may result in chamber-specific differences in myofilament structure and regulation between atria and ventricles. To test this hypothesis, we employed muscle mechanics and X-ray diffraction to investigate functional and structural differences in porcine left atrial (LA) and left ventricular (LV) tissue. Here, we report the first X-ray diffraction study of atrial tissue, demonstrating that under resting conditions, myosin filaments in LA adopted a more ON-like, structurally distinct configuration compared with those in LV. Under contracting conditions, LV generated greater force and exhibited higher sinusoidal stiffness than LA across multiple calcium concentrations. LA showed faster kTR than in LV, with no calcium-dependence, in contrast to the calcium-dependence of kTR seen in LV. Structurally, the distinct myosin head configuration seen in the relaxed LA persisted during contraction. Furthermore, using the troponin inhibitor MYK-7660 to inhibit active contraction, we showed that, unlike LV, LA showed no direct calcium-dependent thick filament activation, reconciling discrepancies between fast rat and slow porcine ventricular myocardium regarding calciums role in thick filament regulation. Altogether, our study reveals that LA myosin filaments adopt a molecular architecture and regulatory mechanism distinct from their LV counterparts, suggesting that myosin filament structure and regulation have evolved differently to meet the unique functional demands of each cardiac chamber. Moreover, atrial disease is often associated with cardiomyopathy-related genetic variants, highlighting the atrial myocardium as an important therapeutic target and understanding atrial-specific regulatory mechanisms provides new insights into therapeutic strategies for atrial diseases.

physiology↗

In silico evaluation of the effects of temperature on the affinity of the SV2C ligand UCB-1A to SV2 isoforms

Synaptic vesicle glycoproteins 2 (SV2) are integral membrane proteins essential for neurotransmitter release and are implicated in neurological disorders including epilepsy and Parkinsons disease. In the attempt to develop a ligand selective for SV2C, and in collaboration with UCB, UCB-F was identified as a potential candidate. However, the affinity of UCB-F to SV2C was found to be temperature dependent, decreasing by about 10-fold from +4 to 37 degrees. UCB1A was subsequently identified as SV2C ligand displaying in vitro a 100-fold selectivity for SV2C compared with SV2A. In this study we investigated whether the binding of UCB-1A to SV2A and SV2C was affected by the temperature. A combination of experimental binding assay data and molecular dynamics (MD) simulations were used. The binding studies revealed that UCB1A affinity for SV2A decreased significantly at 37 {degrees}C compared with 4 {degrees}C, whereas binding to SV2C remained largely unchanged. MD simulations reproduced these observations, namely that ligand RMSD values at 310 K showed that UCB1A binding fluctuated markedly in the SV2A complex, with many trajectories exceeding the 3.0 [A] stability cutoff, whereas UCB1A remained relatively well-anchored in SV2C under the same conditions. Structural analysis showed that, while UCB1A adopts a conserved binding pose across all isoforms stabilized by {pi}- {pi} stacking and a hydrogen bond with Asp, SV2C possesses a unique stabilizing feature. In SV2C, Tyr298 is less exposed to the solvent and engages in a persistent hydrogen bond with Asparagine, a structural feature that reinforces pocket stability and limits temperature-induced destabilization. This interaction is absent in SV2A, consistent with its greater temperature sensitivity. Together, these findings provide a mechanistic explanation for the experimentally observed temperature independence of UCB1A binding to SV2C. More broadly, the results highlight the importance of incorporating physiologically relevant temperatures into SV2 ligand evaluation and demonstrate how combining experiments with simulations can uncover isoform-specific mechanisms of ligand recognition and stability.

biochemistry↗

Discovery and characterization of UCB-1A: the first PET radioligand for imaging synaptic vesicle glycoprotein 2C

The synaptic vesicle glycoprotein 2C (SV2C) is a synaptic protein involved in the regulation of dopamine release. It is expressed in striatum, globus pallidus and substantia nigra, regions involved in the regulation of motor function. Genome-wide association studies, animal model and human brain tissue data indicate a strong link between SV2C and Parkinso[n]s disease, suggesting a potential role of SV2C as synaptic marker for Parkinso[n]s disease. We hypothesize that a positron emission tomography (PET) radioligand for SV2C can serve as imaging marker for Parkinso[n]s disease, enabling early diagnosis and assessment of disease progression. This study was therefore designed to develop a PET radioligand for imaging SV2C. UCB-1A was the lead candidate selected from a library of compounds developed by UCB BioPharma. A translational approach was used, including autoradiography and in vitro binding studies with [3H]UCB-1A, and in vivo PET studies with [11C]UCB-1A in non-human primates (NHPs). The KD of [3H]UCB-1A for rat and human SV2C ranged between 6 and 15 nM, with >100-fold selectivity towards SV2A and SV2B. Specific binding of [3H]UCB-1A in rat and NHP brains was observed in substantia nigra, globus pallidus, striatum and brainstem nuclei, consistent with the expression of SV2C, and was decreased in the striatum of 6-hydroxydopamine-lesioned rats and in the putamen of Parkinson donors. UCB-1A was successfully radiolabelled with 11C and PET studies in NHPs demonstrated that [11C]UCB-1A displays suitable pharmacokinetic properties, a brain distribution consistent with the expression of SV2C and is selective for SV2C. [11C]UCB-1A is the first PET radioligand for in vivo imaging of SV2C and a potential synaptic marker for in vivo studies in Parkinso[n]s disease.

neuroscience↗

Imaging Synaptic Vesicle Protein SV2C with 18F-UCB-F: An In Vitro Autoradiography and In Vivo NHP PET Study

The synaptic vesicle protein SV2C, predominantly found in the basal ganglia, has been associated with Parkinsons disease through genetic studies. It plays a crucial role in regulating dopamine release and has been shown to be disrupted in PD animal models and brain tissues from PD patients. In the context of PD-related synaptopathy, SV2C may serve as a potential imaging target for monitoring disease progression and response to treatment. [18F]UCB-F is a radioligand binding to SV2C developed by UCB. Preliminary autoradiography and PET studies in rats showed that [18F]UCB-F displays a brain distribution consistent with the expression of SV2C in vitro but does not display any specific binding in vivo. This study was therefore designed to further investigate the affinity and selectivity of [18F]UCB-F for SV2C and to examine the in vitro and in vivo properties of the radioligand in non-human primates. In vitro binding studies were performed to measure the affinity of UCB-F to SV2A, SV2B, and SV2C. Insilico modeling was used to assess the binding mode and energy of UCB-F. Autoradiography studies on rat and non-human primate (NHP) brain tissues were performed to confirm that [18F]UCB-F showed similar distribution in rat and NHP tissue. Finally, PET studied in NHPs were performed to examine the in vivo pharmacokinetic properties of [18F]UCB-F. [18F]UCB-F was successfully synthesized from the corresponding precursor with high yield. Autoradiography on brain slices from rats and NHPs demonstrated specific binding of [18F]UCB-F in the pallidum, striatum, substantia nigra, and brainstem, consistent with the known brain expression of SV2C. In NHPs, [18F]UCB-F rapidly crossed the blood-brain barrier, reaching peak uptake values of 2.8 %ID in NHP1 and 2.1 %ID in NHP2 at 4 minutes post-injection. The tracer wasrapidly washed out from the brain, with no clear regional distribution. Radiometabolite analysis revealed the formation of only more polar radiometabolites, with approximately 15% of unchanged radioligand remaining in plasma at 15 minutes post-injection. In vitro and in-silico studies demonstrated that the affinity of [18F]UCB-F decreased by approximately one factor of magnitude with increase of temperature from 4{degrees} to 37{degrees} C. This temperature-related decrease of the affinity for SV2C together with rapid in vivo radiometabolism might explain the discrepancy between in vitro and in vivo performance of [18F]UCB-F. Overall, these findings suggest that [18F]UCB-F is not a suitable PET radioligand for imaging SV2C. Further research is needed to identify alternative candidates with improved in vivo stability and brain retention.

neuroscience↗

The complete genome of the KOLF2.1J reference iPSC line

While induced pluripotent stem cells (iPSCs) have gained popularity in studying neurodegenerative diseases, the heterogeneity of stem cells used across studies impacts cross-study comparison. The iPSC Neurodegenerative Disease Initiative (iNDI) selected the KOLF2.1J cell line and prioritized its use as a reference standard for studying the effects of pathogenic variants on cell biology due to its stability and neutral neurodegenerative disease genetic risk. This cell line, and its derivatives expressing over 100 variants related to Alzheimers disease, Parkinsons disease, and other neurological diseases, are available for academic and industry access. Current genomic data analyses are limited by the use of a human reference genome that does not capture the complete genetic background of a given iPSC line. While in the future this issue may be partially mitigated by the creation of a comprehensive human pangenome, previous work has shown that generating custom genomes is of value both to characterize the variation present and to serve as a more appropriate genomic reference. Here, we generated and characterized a custom complete genome assembly from KOLF2.1J. Mapping of sequencing reads to a personalized diploid assembly results in more comprehensive mapping compared to traditional linear references (i.e GRCh38). In addition, we provide a comprehensive custom gene annotation along with isoform expression and differential methylation analyses across multiple cell types. The assembly and all additional data is browsable and publicly available. This resource will enable more accurate investigation of the KOLF2.1J cell line and any genomics data generated compared to using traditional generalized references, while also serving as a foundational approach for establishing custom reference assemblies for other high-value iPSC lines.

genomics↗

Rapid, accurate long- and short-read mapping to large pangenome graphs with vg Giraffe

1We previously introduced Giraffe, a short-read-to-pangenome graph mapper available in the vg pangenomics toolkit. Giraffe was fast and accurate for mapping short reads to human-scale pangenomes, but struggled with long reads. Long reads present a unique challenge to pangenome mapping algorithms due to their length and error profile, which allow them to take more topologically complex paths through the pangenome graph and increase the possible search space for the algorithm. We present updates to Giraffe that allow it to quickly and accurately map long reads to pangenome graphs. For both short and long reads, Giraffe mapping to a pangenome containing data from more than 450 human haplotypes, generated by the Human Pangenome Reference Consortium, is comparable in speed to linear mappers to human reference genomes; Giraffe is also over an order of magnitude faster than GraphAligner, the current state-of-the-art long-read-to-pangenome mapper. Its alignments produce similar or improved small and structural variant calling results, compared to those from commonly used graph-based and linear mappers. We additionally demonstrate using Giraffes long read alignments in a pangenome-guided assembly workflow, which is capable of producing more contiguous local assemblies than Hifiasm in our test regions.

bioinformatics↗

ATG2A engages Rab1a and ARFGAP1 positive membranes during autophagosome biogenesis

Autophagosomes form from seed membranes that expand through bulk-lipid transport via the bridge-like lipid transporter ATG2. The origins of the seed membranes and their relationship to the lipid transport machinery are poorly understood. Using proximity labeling and a variety of fluorescence microscopy techniques, we show that ATG2A localizes to extra-Golgi ARFGAP1 puncta during autophagosome biogenesis. ARFGAP1 itself is dispensable during macroautophagy, but among other proteins associating to these membranes, we find that RAB1 is essential. ATG2A co-immunoprecipitates strongly, albeit indirectly, with RAB1A, and siRNA-mediated depletion of RAB1A/B blocks autophagy downstream of LC3B lipidation, similar to ATG2A depletion. Further, when either autophagosome formation or the early secretory pathway is perturbed, ARFGAP1 and RAB1A accumulate at ectopic locations with autophagic machinery. Our results indicate that ATG2A engages a RAB1A complex on select early secretory membranes in support of autophagosome biogenesis. Significance StatementThis study expounds upon the role of early secretory membranes in autophagosome biogenesis. The authors demonstrate that RAB1/ARFGAP1-positive membranes are essential to autophagy and are recruited to the phagophore assembly site at an early step of autophagosome biogenesis. These membranes interact with the bridge-like lipid transport protein ATG2A and are positive for LC3B and WIPI2, suggesting that RAB1 membranes are a direct source for autophagosome formation.

cell biology↗

PET imaging of an antisense oligonucleotide in the living non-human primate brain using click chemistry

Determination of a drugs biodistribution is critical to ensure it reaches the target tissue of interest. This is particularly challenging in the brain where invasive sampling methods may not be possible. Here, a pretargeted imaging methodology is disclosed that utilizes bioorthogonal click chemistry to determine the distribution of an antisense oligonucleotide in the living brain following intrathecal dosing. A novel PET tracer, [18F]BIO-687, bearing a click-reactive trans-cyclooctene (TCO) was discovered and tested in conjunction with a Malat1 antisense oligonucleotide (ASO) conjugated with a methyltetrazine (MeTz). PET imaging in rats demonstrated that the tracer possesses good kinetic properties for CNS imaging and can react to form a covalent linkage with high specificity to the MeTz-conjugated-ASO in vivo. Further, the amount of tracer reacted by cycloaddition with the Tz was determined to be dependent on the concentration of ASO-MeTz in tissue, as determined through comparison of the imaging signal with the LC-MS of the tissue homogenate. The system was evaluated in cynomolgus monkeys, with PET imaging showing favorable tracer kinetics and specific binding to the ASO in vivo. These results demonstrate that the tracer [18F]BIO-687 can image intrathecally-delivered ASO distribution in the brain, and future studies should leverage this technology to evaluate ASO distribution in human subjects to study distribution. One Sentence SummaryDistribution of an intrathecally administered antisense oligonucleotide can be imaged using a pretargeted approach in the living brains of non-human primates.

neuroscience↗

E2F1 Suppresses EBV Lytic Reactivation through Cellular and Viral Transcriptional Networks

Latent EBV infection is causally associated with various B-cell malignancies, while periodic lytic-cycle replication is essential for sustaining viral progeny. Lytic cycle induction represents a promising therapeutic strategy for EBV-associated neoplasms. Therefore, uncovering the mechanisms that regulate EBV lytic-cycle reactivation is pivotal for understanding viral pathogenesis and advancing novel therapies. Our genome-wide transcriptomic analysis reveals that E2F1 expression is transcriptionally activated during EBV latent infection in B-lymphocytes but significantly suppressed during lytic cycle reactivation. While ectopic E2F1 expression suppresses lytic replication, E2F1 depletion markedly accelerates this process. Mechanistically, we establish that E2F1 and the lytic transactivator BZLF1 form a negative transcriptional feedback loop, tightly controlling viral lytic replication. Furthermore, E2F1 positively regulates c-Myc expression and together they repress the leaky BZLF1 expression during latency. Importantly, c-Myc does not influence E2F1 expression, nor does BZLF1 modulate c-Myc transcription, underlining a distinct regulatory hierarchy. In sum, our findings reveal that EBV tightly controls the latent-to-lytic switch through precise regulation of E2F1 expression, positioning E2F1 as a pivotal regulator of both cellular and viral gene expression. SynopsisEBV coordinates the latent-to-lytic switch by sensing E2F1 abundance, which acts as a crucial transcriptional regulator of both cellular and viral gene expressions. O_LIDuring EBV latent infection, E2F1 promotes c-Myc transcription, and together they suppress EBV lytic cycle transactivator BZLF1 expression. C_LIO_LIE2F1 and BZLF1 form a negative feedback loop in order to control each others transcriptions. C_LIO_LIBZLF1-driven controlled E2F1 expression successively inhibits c-Myc level, thereby stimulating EBV lytic cycle reactivation. C_LIO_LIBZLF1 does not regulate c-Myc, nor does c-Myc reciprocally regulate E2F1, emphasizing a unidirectional regulatory hierarchy. C_LI

cancer biology↗

Mutualism Destabilizes Communities, but Competition Pays the Price

A classic result in theoretical ecology states that an increase in the proportion of cooperative interactions in unstructured ecological communities leads to a loss of stability to external perturbations. However, the fate and composition of the species that constitute an unstable ecological community following such perturbations remains relatively unexplored. In this paper, we use an individual-based model to study the population dynamics of unstructured communities following external perturbations to species abundances. We find that while increasing the number of cooperative interactions does indeed increase the probability that a community will experience an extinction following a perturbation, the entire community is rarely wiped out following a perturbation. Instead, only a subset of the ecological community is driven to extinction, and the species that go extinct are more likely to be those engaged in a greater number of competitive interactions. Thus, the resultant community formed after a perturbation has a higher proportion of cooperative interactions than the original community. We show that this result can be explained by studying the dynamics of the species engaged in the highest number of competitive interactions: After an external perturbation, those species that compete with such a top competitor are more likely to go extinct than expected by chance alone, whereas those that are engaged in cooperative interactions with such a species are less likely to go extinct than expected by chance alone. Our results provide a potential explanation for the ubiquity of cooperative interactions in nature despite the known negative effects of cooperation on community stability.

ecology↗

Human disease-specific cell signatures in non-lesional tissue in Multiple Sclerosis detected by single-cell and spatial transcriptomics

Recent investigations of cell type changes in Multiple Sclerosis (MS) using single-cell profiling methods have focused on active lesional and peri-lesional brain tissue, and have implicated a number of peripheral and central nervous system cell types. However, an important question is the extent to which so-called "normal-appearing" non-lesional tissue in individuals with MS accumulate changes over the lifespan. Here, we compared post-mortem non-lesional brain tissue from donors with a pathological or clinical diagnosis of MS from the Religious Orders Study or Rush Memory and Aging Project (ROSMAP) cohorts to age- and sex-matched brains from persons without MS (controls). We profiled three brain regions using single-nucleus RNA-seq: dorsolateral prefrontal cortex (DLPFC), normal appearing white matter (NAWM) and the pulvinar in thalamus (PULV), from 15 control individuals, 8 individuals with MS, and 5 individuals with other detrimental pathologies accompanied by demyelination, resulting in a total of 78 samples. We identified region- and cell type-specific differences in non-lesional samples from individuals diagnosed with MS and/or exhibiting secondary demyelination with other neurological conditions, as compared to control donors. These differences included lower proportions of oligodendrocytes with expression of myelination related genes MOBP, MBP, PLP1, as well as higher proportions of CRYAB+ oligodendrocytes in all three brain regions. Among microglial signatures, we identified subgroups that were higher in both demyelination (TMEM163+/ERC2+), as well as those that were specifically higher in MS donors (HIF1A+/SPP1+) and specifically in donors with secondary demyelination (SOCS6+/MYO1E+), in both white and grey matter. To validate our findings, we generated Visium spatial transcriptomics data on matched tissue from 13 donors, and recapitulated our observations of gene expression differences in oligodendrocytes and microglia. Finally, we show that some of the differences observed between control and MS donors in NAWM mirror those previously reported between control WM and active lesions in MS donors. Overall, our investigation sheds additional light on cell type- and disease-specific differences present even in non-lesional white and grey matter tissue, highlighting widespread cellular signatures that may be associated with downstream pathological changes.

neuroscience↗

Differential Carbonic Anhydrase Activities Control EBV-Induced B-Cell Transformation and Lytic Cycle Reactivation

Epstein-Barr virus (EBV) contributes to [~]1% of all human cancers including several B-cell neoplasms. A characteristic feature of EBV life cycle is its ability to transform metabolically quiescent B-lymphocytes into hyperproliferating B-cell blasts with the establishment of viral latency, while intermittent lytic cycle induction is necessary for the production of progeny virus. Our RNA-Seq analyses of both latently infected naive B-lymphocytes and transformed B-lymphocytes upon lytic cycle replication indicate a contrasting expression pattern of a membrane-associated carbonic anhydrase isoform CA9, an essential component for maintaining cell acid-base homeostasis. We show that while CA9 expression is transcriptionally activated during latent infection model, lytic cycle replication restrains its expression. Pharmacological inhibition of CA-activity using specific inhibitors retards EBV induced B-cell transformation, inhibits B-cells outgrowth and colony formation ability of transformed B-lymphocytes through lowering the intracellular pH, induction of cell apoptosis and facilitating degradation of CA9 transcripts. Reanalyses of ChIP-Seq data along with utilization of EBNA2 knockout virus and sh-RNA mediated knockdown of CA9 expression we further demonstrate that EBNA2 mediated CA9 transcriptional activation is essential for EBV latently infected B-cell survival. In contrast, during lytic cycle reactivation CA9 expression is transcriptionally suppressed by the key EBV lytic cycle transactivator, BZLF1 through its transactivation domain. Overall, our study highlights the dynamic alterations of CA9 expression and its activity in regulating pH homeostasis act as one of the major drivers for EBV induced B-cell transformation and subsequent B-cell lymphomagenesis.

microbiology↗

Myosin in autoinhibited off state(s), stabilized by mavacamten, can be recruited via inotropic effectors

Mavacamten is a novel, FDA-approved, small molecule therapeutic designed to regulate cardiac function by selectively but reversibly inhibiting the enzymatic activity of myosin. It shifts myosin towards ordered off states close to the thick filament backbone. It remains unresolved whether mavacamten permanently sequesters these myosin heads in the off state(s) or whether these heads can be recruited in response to physiological stimuli when required to boost cardiac output. We show that cardiac myosins stabilized in these off state(s) by mavacamten are recruitable by Ca2+, increased heart rate, stretch, and {beta}-adrenergic ({beta}-AR) stimulation, all known physiological inotropic effectors. At the molecular level, we show that, in presence of mavacamten, Ca2+ increases myosin ATPase activity by shifting myosin heads from the reserve super-relaxed (SRX) state to the active disordered relaxed (DRX) state. At the myofilament level, both Ca2+ and passive lengthening can shift ordered off myosin heads from positions close to the thick filament backbone to disordered on states closer to the thin filaments in the presence of mavacamten. In isolated rat cardiomyocytes, increased stimulation rates enhanced shortening fraction in mavacamten-treated cells. This observation was confirmed in vivo in telemetered rats, where left-ventricular dP/dtmax, an index of inotropy, increased with heart rate in mavacamten treated animals. Finally, we show that {beta}-AR stimulation in vivo increases left-ventricular function and stroke volume in the setting of mavacamten. Our data demonstrate that the mavacamten-promoted off states of myosin in the thick filament are activable, at least partially, thus leading to preservation of cardiac reserve mechanisms. Significance statementMavacamten is the first myosin-targeted small molecule inhibitor approved by the FDA to treat obstructive hypertrophic cardiomyopathy by attenuating myocardial hyperdynamic contraction. The recruitment of cardiac contractility is, however, vital to ensure sufficient cardiac output during increased physiological demand. Here we show that major inotropic effectors are at least partially preserved in the setting of mavacamten, resulting in maintenance of cardiac reserve mechanisms. These results not only suggest an alternative mechanistic explanation, beyond mere LV outflow tract obstruction removal, for the clinically observed increase in peak oxygen uptake with exercise in HCM patients receiving mavacamten, but also lay the groundwork for a potential methodology to investigate the sarcomeric basis of chronotropic incompetence in disease states to motivate new therapeutic interventions.

physiology↗

A small-molecule myosin inhibitor as a targeted multi-stage antimalarial

Malaria is a devastating disease that resulted in an estimated 627,000 deaths in 2020. About 80% of those deaths were among children under the age of five. Our approach is to develop small molecule inhibitors against cytoskeletal targets that are vital components of parasite function, essential at multiple stages of parasite infection, can be targeted with high specificity, and are highly druggable. Here we describe KNX-115, which inhibits purified Plasmodium falciparum myosin A (PfMyoA) actin-activated ATPase with a potency in the 10s of nanomolar range and >50-fold selectivity against cardiac, skeletal, and smooth muscle myosins. KNX-115 inhibits the blood and liver stages of Plasmodium with an EC50 of about 100 nanomolar, with negligible liver cell toxicity. In addition, KNX-115 inhibits sporozoite cell traversal and blocks the gametocyte to oocyst conversion in the mosquito. KNX-115 displays a similar killing profile to pyrimethamine and parasites are totally killed after 96 hours of treatment. In line with its novel mechanism of action, KNX-115 is equally effective at inhibiting a panel of Plasmodium strains resistant to experimental and marketed antimalarials. In vitro evolution data likely suggests a refractory potential of KNX-115 in developing parasite resistance.

pharmacology and toxicology↗

ATG9 vesicles comprise the seed membrane of mammalian autophagosomes

During autophagosome biogenesis, the incorporation of transmembrane proteins into the expanding phagophore is not readily observed. In addition, the membrane surface area of the organelle expands rapidly, while the volume of the autophagosome is kept low. Several recent studies have suggested a model of membrane expansion that explains how these attributes are maintained. The autophagosome expands predominantly through the direct protein-mediated transfer of lipids through the lipid transfer protein ATG2. As these lipids are only introduced into the cytoplasmic-facing leaflet of the expanding phagophore, full membrane growth also requires lipid scramblase activity. ATG9 has been demonstrated to harbor scramblase activity and is essential to autophagosome formation, however if and when it is integrated into mammalian autophagosomes remains unclear. Here we show that in the absence of lipid transport, ATG9 vesicles are already fully competent to collect proteins normally found on mature autophagosomes, including LC3-II. Further, through the novel use of styrene-maleic acid lipid particles as a nanoscale interrogation of protein organization on intact membranes, we show that ATG9 is fully integrated in the same membranes as LC3-II, even on maturing autophagosomes. The ratios of these two proteins at different stages of maturation demonstrate that ATG9 proteins are not continuously integrated, but rather are present on the seed vesicles only and become diluted in the rapidly expanding autophagosome membrane. Thus, ATG9 vesicles are the seed membrane from which mammalian autophagosomes form.

cell biology↗

Studying the age of onset and detection of Chronic Myeloid Leukemia using a three-stage stochastic model

Chronic Myeloid Leukemia (CML) is a biphasic malignant clonal disorder that progresses, first with a chronic phase, where the cells have enhanced proliferation only, and then to a blast phase, where the cells have the ability of self-renewal. It is well-recognized that the Philadelphia chromosome (which contains the BCR-ABL fusion gene) is the "hallmark of CML". However, empirical studies have shown that the mere presence of BCR-ABL may not be a sufficient condition for the development of CML, and further modifications related to tumor suppressors may be necessary. Accordingly, we develop a three-mutation stochastic model of CML progression, with the three stages corresponding to the non-malignant cells with BCR-ABL presence, the malignant cells in the chronic phase and the malignant cells in the blast phase. We demonstrate that the model predictions agree with age incidence data from the United States. Finally, we develop a framework for the retrospective estimation of the time of onset of malignancy, from the time of detection of the cancer.

systems biology↗

Assessment of local potato cultivars found in Cis-Himalayan region of West Bengal through morphology and biochemical profiling

Potato is a major global food crop grown for tubers (underground storage stems) that are high in carbohydrates, minerals, and vitamins. The presence of anthocyanins in tuber skin and flesh can have a significant impact on customer preferences. Potatoes are also high in resistant starches, which have a variety of health benefits, including enhanced fullness, cholesterol reduction, and a reduced risk of colon cancer etc. In West Bengal, diabetes is prevalent. Similarly, according to ICMR studies, colon cancer affects 8.9% of all cancer patients in West Bengal cancer which is caused by ill eating habits or the consumption of foods that are low in antioxidants. 5-10% of colon cancers are genetically caused, while the remainder are caused by poor eating habits or the consumption of foods that are low in antioxidants. To address these issues, one strategy is to eat foods with a low glycemic index and high antioxidant content as a staple food. The tuber tissues of the potato (S. tuberosum L.) accumulate various quantities of anthocyanins, which are commonly consumed around the world. Anthocyanins are pigments that range in colour from red to purple and are found throughout the plant kingdom. Anthocyanins are powerful antioxidants that are water soluble. The red skin potato is well-known among the general public. The epidermal layer contains a high quantity of red anthocyanins, which causes the skin to be red. With this background the present study has been undertaken to address the following objectives morphological (quantitative and qualitative traits) and biochemical characterization of local potato cultivars and identification of most stable genotypes based on the anthocyanin content and tuber yield of local potato cultivars. For our recent research 14 local potato cultivar from North Bengal were collected and evaluated for DUS characterization showed wide range of variability with respect to different phenotypic variants of ten characters. Overall predominant DUS characters of local potato cultivars found in North Bengal are medium sized apical length of sprout, short stem height, compact foliage structure, open leaf structure, ovate lanceolate type leaflet shape, purple leaf sprout predominant colour, spherical leaf sprout shape, medium intensity of anthicyanin coloraton at the base, light intensity of anthicyanin coloraton at the tip and weak nature of light sprout pubescence base. The results of the present investigation suggest that local potato cultivars collected from the northern part of West Bengal showed a high level of genetic variation. The differences between genotypes were highly significant at the 1% and 5% alpha level for all characters like tuber yield, length and weight of tuber, length of sprout, chlorophyll content, canopy temperature, and number of tuber per plant, according to the analysis of variance. Three quantitative traits namely tuber yield, tuber breadth, tuber length played major role in the genetic variance. Anthocyanin content had more contribution to diversify the local potato cultures according to biochemical characterization AMMI analysis suggested that Jalpai from CoochBehar is the most stable potato cultivars in respect to anthocyanin content and tuber yield per plant.

plant biology↗