Vaccine priming of rare HIV broadly neutralizing antibody precursors in nonhuman primates.

Germline-targeting immunogens hold promise for initiating the induction of broadly neutralizing antibodies (bnAbs) to HIV and other pathogens. However, antibody-antigen recognition is typically dominated by heavy chain complementarity determining region 3 (HCDR3) interactions, and vaccine priming of HCDR3-dominant bnAbs by germline-targeting immunogens has not been demonstrated in humans or outbred animals. In this work, immunization with N332-GT5, an HIV envelope trimer designed to target precursors of the HCDR3-dominant bnAb BG18, primed bnAb-precursor B cells in eight of eight rhesus macaques to substantial frequencies and with diverse lineages in germinal center and memory B cells. We confirmed bnAb-mimicking, HCDR3-dominant, trimer-binding interactions with cryo-electron microscopy. Our results demonstrate proof of principle for HCDR3-dominant bnAb-precursor priming in outbred animals and suggest that N332-GT5 holds promise for the induction of similar responses in humans.

Vaccination induces broadly neutralizing antibody precursors to HIV gp41.

A key barrier to the development of vaccines that induce broadly neutralizing antibodies (bnAbs) against human immunodeficiency virus (HIV) and other viruses of high antigenic diversity is the design of priming immunogens that induce rare bnAb-precursor B cells. The high neutralization breadth of the HIV bnAb 10E8 makes elicitation of 10E8-class bnAbs desirable; however, the recessed epitope within gp41 makes envelope trimers poor priming immunogens and requires that 10E8-class bnAbs possess a long heavy chain complementarity determining region 3 (HCDR3) with a specific binding motif. We developed germline-targeting epitope scaffolds with affinity for 10E8-class precursors and engineered nanoparticles for multivalent display. Scaffolds exhibited epitope structural mimicry and bound bnAb-precursor human naive B cells in ex vivo screens, protein nanoparticles induced bnAb-precursor responses in stringent mouse models and rhesus macaques, and mRNA-encoded nanoparticles triggered similar responses in mice. Thus, germline-targeting epitope scaffold nanoparticles can elicit rare bnAb-precursor B cells with predefined binding specificities and HCDR3 features.

Digger: directed annotation of immunoglobulin and T cell receptor V, D, and J gene sequences and assemblies.

SUMMARY: Knowledge of immunoglobulin and T cell receptor encoding genes is derived from high-quality genomic sequencing. High-throughput sequencing is delivering large volumes of data, and precise, high-throughput approaches to annotation are needed. Digger is an automated tool that identifies coding and regulatory regions of these genes, with results comparable to those obtained by current expert curational methods. AVAILABILITY AND IMPLEMENTATION: Digger is published under open source license at https://github.com/williamdlees/Digger and is available as a Python package and a Docker container.

The SET7 protein of Leishmania donovani moderates the parasite's response to a hostile oxidative environment.

SET domain proteins methylate specific lysines on proteins, triggering stimulation or repression of downstream processes. Twenty-nine SET domain proteins have been identified in Leishmania donovani through sequence annotations. This study initiates the first investigation into these proteins. We find LdSET7 is predominantly cytosolic. Although not essential, set7 deletion slows down promastigote growth and hypersensitizes the parasite to hydroxyurea-induced G1/S arrest. Intriguingly, set7-nulls survive more proficiently than set7+/+ parasites within host macrophages, suggesting that LdSET7 moderates parasite response to the inhospitable intracellular environment. set7-null in vitro promastigote cultures are highly tolerant to hydrogen peroxide (H2O2)-induced stress, reflected in their growth pattern, and no detectable DNA damage at H2O2 concentrations tested. This is linked to reactive oxygen species levels remaining virtually unperturbed in set7-nulls in response to H2O2 exposure, contrasting to increased reactive oxygen species in set7+/+ cells under similar conditions. In analyzing the cell's ability to scavenge hydroperoxides, we find peroxidase activity is not upregulated in response to H2O2 exposure in set7-nulls. Rather, constitutive basal levels of peroxidase activity are significantly higher in these cells, implicating this to be a factor contributing to the parasite's high tolerance to H2O2. Higher levels of peroxidase activity in set7-nulls are coupled to upregulation of tryparedoxin peroxidase transcripts. Rescue experiments using an LdSET7 mutant suggest that LdSET7 methylation activity is critical to the modulation of the cell's response to oxidative environment. Thus, LdSET7 tunes the parasite's behavior within host cells, enabling the establishment and persistence of infection without eradicating the host cell population it needs for survival.

The extremophile Picrophilus torridus carries a DNA adenine methylase M.PtoI that is part of a Type I restriction-modification system.

DNA methylation events mediated by orphan methyltransferases modulate various cellular processes like replication, repair and transcription. Bacteria and archaea also harbor DNA methyltransferases that are part of restriction-modification systems, which serve to protect the host genome from being cleaved by the cognate restriction enzyme. While DNA methylation has been exhaustively investigated in bacteria it remains poorly understood in archaea. Picrophilus torridus is a euryarchaeon that can thrive under conditions of extremely low pH (0.7), and thus far no reports have been published regarding DNA methylation in this extremophile. This study reports the first experimentation examining DNA methylation in P. torridus. We find the genome to carry methylated adenine (m6A) but not methylated cytosine (m5C) residues. The m6A modification is absent at GATC sites, indicating the absence of an active Dam methylase even though the dam gene has been annotated in the genome sequence. Two other methylases have also been annotated in the P. torridus genome sequence. One of these is a part of a Type I restriction-modification system. Considering that all Type I modification methylases characterized to date target adenine residues, the modification methylase of this Type I system has been examined. The genes encoding the S subunit (that is responsible for DNA recognition) and M subunit (that is responsible for DNA methylation) have been cloned and the recombinant protein purified from E.coli, and regions involved in M-S interactions have been identified. The M.PtoI enzyme harbors all the motifs that typify Type I modification methylases, and displays robust adenine methylation in in vitro assays under a variety of conditions. Interestingly, magnesium is essential for enzyme activity. The enzyme displays substrate inhibition at higher concentrations of AdoMet. Mutational analyses reveal that Motif I plays a role in AdoMet binding, and Motif IV is critical for methylation activity. The data presented here lays the foundation for further research in the area of DNA methylation and restriction-modification research in this most unusual microorganism.

Settlement suitability analysis of a riverine floodplain in the perspective of GIS-based multicriteria decision analysis.

Riverine floodplains are highly dynamic and the most vulnerable space on Earth particularly in flat alluvial plains of major river systems. Suitable site selection for sustainable human settlements in active floodplain areas is a critical task for decision-makers in terms of quality of lithology, ecologically fragile landmass, climate-induced hazards, immense population pressure, and environmental conservation issues. This research introduces a methodology for settlement suitability zone (SSZ) that employs GIS-based multi-criteria decision-making (MCDM) techniques. As a case study, an altered hydrological regime of the lower Ganga riparian zone was chosen to identify the SSZ as these areas are the most susceptible to riverine hazards. Twelve significant variables reflecting on topography, climate, landscape, and environment have been selected in the multi-criteria evaluation platform. The CRiteria Importance Through Intercriteria Correlation (CRITIC) method is adopted to specify the weights of the criteria and utilize an inverse distance-weighted (IDW) spatial interpolation technique to generate an SSZ map in a GIS environment. The study zone is spatially quantified into five categories, from unsuitable to high-suitable with a natural breaks (Jenks) classification method. Subsequently, the final results are validated through a receiver operating characteristics (ROC) curve using randomly selected 56 hazard-exposed location points. The outcome revealed that 8.45% of the riparian area falls under unsuitable, 21.87% under low-suitable, and 33.27% under moderate-suitable for locating human settlements. The generally suitable and high-suitable categories account for 36.40% of the total study zone. A spatial sensitivity analysis is also applied to gauge the influence of each parameter on the MCDM outcomes. The SSZ mapping results from this study can help local authorities to plan for sustainable settlement development in environmentally fragile areas.

Correction: DNA replication protein Cdc45 directly interacts with PCNA via its PIP box in Leishmania donovani and the Cdc45 PIP box is essential for cell survival.

[This corrects the article DOI: 10.1371/journal.ppat.1008190.].

Automated credit assessment framework using ETL process and machine learning.

In the current business scenario, real-time analysis of enterprise data through Business Intelligence (BI) is crucial for supporting operational activities and taking any strategic decision. The automated ETL (extraction, transformation, and load) process ensures data ingestion into the data warehouse in near real-time, and insights are generated through the BI process based on real-time data. In this paper, we have concentrated on automated credit risk assessment in the financial domain based on the machine learning approach. The machine learning-based classification techniques can furnish a self-regulating process to categorize data. Establishing an automated credit decision-making system helps the lending institution to manage the risks, increase operational efficiency and comply with regulators. In this paper, an empirical approach is taken for credit risk assessment using logistic regression and neural network classification method in compliance with Basel II standards. Here, Basel II standards are adopted to calculate the expected loss. The required data integration for building machine learning models is done through an automated ETL process. We have concluded this research work by evaluating this new methodology for credit risk assessment.

Histone Modifications and Other Facets of Epigenetic Regulation in Trypanosomatids: Leaving Their Mark.

Histone posttranslational modifications (PTMs) modulate several eukaryotic cellular processes, including transcription, replication, and repair. Vast arrays of modifications have been identified in conventional eukaryotes over the last 20 to 25 years. While initial studies uncovered these primarily on histone tails, multiple modifications were subsequently found on the central globular domains as well. Histones are evolutionarily conserved across eukaryotes, and a large number of their PTMs and the functional relevance of these PTMs are largely conserved. Trypanosomatids, however, are early diverging eukaryotes. Although possessing all four canonical histones as well as several variants, their sequences diverge from those of other eukaryotes, particularly in the tails. Consequently, the modifications they carry also vary. Initial analyses almost 15 years ago suggested that trypanosomatids possessed a smaller collection of histone modifications. However, exhaustive high resolution mass spectrometry analyses in the last few years have overturned this belief, and it is now evident that the "histone code" proposed by Allis and coworkers in the early years of this century is as complex in these organisms as in other eukaryotes. Trypanosomatids cause several diseases, and the members of this group of organisms have varied lifestyles, evolving diverse mechanisms to evade the host immune system, some of which have been found to be principally controlled by epigenetic mechanisms. This minireview aims to acquaint the reader with the impact of histone PTMs on trypanosomatid cellular processes, as well as other facets of trypanosomatid epigenetic regulation, including the influence of three-dimensional (3D) genome architecture, and discusses avenues for future investigations.

DNA replication protein Cdc45 directly interacts with PCNA via its PIP box in Leishmania donovani and the Cdc45 PIP box is essential for cell survival.

DNA replication protein Cdc45 is an integral part of the eukaryotic replicative helicase whose other components are the Mcm2-7 core, and GINS. We identified a PIP box motif in Leishmania donovani Cdc45. This motif is typically linked to interaction with the eukaryotic clamp proliferating cell nuclear antigen (PCNA). The homotrimeric PCNA can potentially bind upto three different proteins simultaneously via a loop region present in each monomer. Multiple binding partners have been identified from among the replication machinery in other eukaryotes, and the concerted /sequential binding of these partners are central to the fidelity of the replication process. Though conserved in Cdc45 across Leishmania species and Trypanosoma cruzi, the PIP box is absent in Trypanosoma brucei Cdc45. Here we investigate the possibility of Cdc45-PCNA interaction and the role of such an interaction in the in vivo context. Having confirmed the importance of Cdc45 in Leishmania DNA replication we establish that Cdc45 and PCNA interact stably in whole cell extracts, also interacting with each other directly in vitro. The interaction is mediated via the Cdc45 PIP box. This PIP box is essential for Leishmania survival. The importance of the Cdc45 PIP box is also examined in Schizosaccharomyces pombe, and it is found to be essential for cell survival here as well. Our results implicate a role for the Leishmania Cdc45 PIP box in recruiting or stabilizing PCNA on chromatin. The Cdc45-PCNA interaction might help tether PCNA and associated replicative DNA polymerase to the DNA template, thus facilitating replication fork elongation. Though multiple replication proteins that associate with PCNA have been identified in other eukaryotes, this is the first report demonstrating a direct interaction between Cdc45 and PCNA, and while our analysis suggests the interaction may not occur in human cells, it indicates that it may not be confined to trypanosomatids.

Sustainability assessment for the transportation environment of Darjeeling, India.

Darjeeling is an important tourist hill town of West Bengal, India. It suffers from an acute problem of transportation, particularly during its peak tourist seasons due to limited road space, inadequate public transport facilities and indiscriminate use of automobiles. This hill town was originally designed for a population of 10,000, but over the years, it has come face-to-face with rapid urbanization, a rising population of both tourists and residents and intensifying motor vehicle usage. These factors together are posing a threat to its transport environment. This study identifies the Sustainable Transport Indicators (STIs) available in the existing literature to identify the critical stretches using Analytical Hierarchy Process (AHP) based on experts' consensus. It was found that the experts placed emphasis on the mobility of the town, talking about vehicular impact on air pollution and encroachment of roads as the main issues affecting the sustainability of the transport environment. Thereafter, policy-level interventions have been suggested in accordance with the identified sustainability issues. We trust that other tourist hill towns with issues similar to Darjeeling could easily emulate the study methodology to assess their transport environment sustainability, or replicate on the lines of the recommended policy interventions.

Cell cycle stage-specific transcriptional activation of cyclins mediated by HAT2-dependent H4K10 acetylation of promoters in Leishmania donovani.

Chromatin modifications affect several processes. In investigating the Leishmania donovani histone acetyltransferase HAT2, using in vitro biochemical assays and HAT2-heterozygous genomic knockout we found the constitutively nuclear HAT2 acetylated histone H4K10 in vitro and in vivo. HAT2 was essential. HAT2-depleted cells displayed growth and cell cycle defects, and poor survival in host cells. Real time PCR and DNA microarray analyses, as well as rescue experiments, revealed that downregulation of cyclins CYC4 and CYC9 were responsible for S phase and G2/M defects of HAT2-depleted cells respectively. Leishmania genes are arranged in unidirectional clusters, and clustered genes are coordinately transcribed as long polycistronic units, typically from divergent strand switch regions (dSSRs) which initiate transcription bidirectionally on opposite strands. In investigating the mechanism by which CYC4 and CYC9 expression levels are reduced in HAT2-depleted cells without other genes in their polycistronic transcription units being coordinately downregulated, we found using reporter assays that CYC4 and CYC9 have their own specific promoters. Chromatin immunoprecipitation assays with H4acetylK10 antibodies and real time PCR analyses of RNA suggested these gene-specific promoters were activated in cell cycle-dependent manner. Nuclear run-on analyses confirmed that CYC4 and CYC9 were transcriptionally activated from their own promoters at specific cell cycle stages. Thus, there are two tiers of gene regulation. Transcription of polycistronic units primarily initiates at dSSRs, and this most likely occurs constitutively. A subset of genes have their own promoters, at least some of which are activated in a cell-cycle dependent manner. This second tier of regulation is more sensitive to H4K10 acetylation levels, resulting in downregulation of expression in HAT2-depleted cells. This report presents the first data pointing to cell cycle-specific activation of promoters in trypanosomatids, thus uncovering new facets of gene regulation in this parasite family.

Histone acetyltransferase HAT4 modulates navigation across G2/M and re-entry into G1 in Leishmania donovani.

Histone acetyltransferases impact multiple processes. This study investigates the role of histone acetyltransferase HAT4 in Leishmania donovani. Though HAT4 was dispensable for survival, its elimination decreased cell viability and caused cell cycle defects, with HAT4-nulls experiencing an unusually long G2/M. Survival of HAT4-nulls in macrophages was also substantially compromised. DNA microarray analysis revealed that HAT4 modestly regulated the expression of only a select number of genes, thus not being a major modulator of global gene expression. Significantly, cdc20 was among the downregulated genes. To ascertain if decreased expression of cdc20 was responsible for HAT4-null growth and cell cycle defects we expressed LdCdc20 ectopically in HAT4-nulls. We found this to alleviate the aberrant growth and cell cycle progression patterns displayed by HAT4-nulls, with cells navigating G2/M phase and re-entering G1 phase smoothly. HAT4-nulls expressing LdCdc20 ectopically showed survival rates comparable to wild type within macrophages, suggesting that G2/M defects were responsible for poor survival of HAT4-nulls within host cells also. These are the first data analyzing the in vivo functional role of HAT4 in any trypanosomatid. Our results directly demonstrate for the first time a role for Cdc20 in regulating trypanosomatid G2/M events, opening avenues for further research in this area.

Incidence of Lettuce mosaic virus in lettuce and its detection by polyclonal antibodies produced against recombinant coat protein expressed in Escherichia coli.

Lettuce mosaic virus (LMV), a member of the genus Potyvirus of family Potyviridae, causes mosaic disease in lettuce has recently been identified in India. The virus is seed borne and secondary infection occurs through aphids. To ensure virus freedom in seeds it is important to develop diagnostic tools, for serological methods the production of polyclonal antibodies is a prerequisite. The coat protein (CP) gene of LMV was amplified, cloned and expressed using pET-28a vector in Escherichia coli BL21DE3 competent cells. The LMV CP was expressed as a fusion protein containing a fragment of the E. coli His tag. The LMV CP/His protein reacted positively with a commercial antiserum against LMV in an immunoblot assay. Polyclonal antibodies purified from serum of rabbits immunized with the fusion protein gave positive results when LMV infected lettuce (Lactuca sativa) was tested at 1:1000 dilution in PTA-ELISA. These were used for specific detection of LMV in screening lettuce accessions. The efficacy of the raised polyclonal antiserum was high and it can be utilized in quarantine and clean seed production.

HAT3-mediated acetylation of PCNA precedes PCNA monoubiquitination following exposure to UV radiation in Leishmania donovani.

Histone modifications impact various processes. In examining histone acetyltranferase HAT3 of Leishmania donovani, we find elimination of HAT3 causes decreased cell viability due to defects in histone deposition, and aberrant cell cycle progression pattern. HAT3 associates with proliferating cell nuclear antigen (PCNA), helping load PCNA onto chromatin in proliferating cells. HAT3-nulls show heightened sensitivity to UV radiation. Following UV exposure, PCNA cycles off/on chromatin only in cells expressing HAT3. Inhibition of the ubiquitin-proteasome pathway prior to UV exposure allows accumulation of chromatin-bound PCNA, and reveals that HAT3-nulls are deficient in PCNA monoubiquitination as well as polyubiquitination. While poor monoubiquitination of PCNA may adversely affect translesion DNA synthesis-based repair processes, polyubiquitination deficiencies may result in continued retention of chromatin-bound PCNA, leading to genomic instability. On suppressing the proteasome pathway we also find that HAT3 mediates PCNA acetylation in response to UV. HAT3-mediated PCNA acetylation may serve as a flag for PCNA ubiquitination, thus aiding DNA repair. While PCNA acetylation has previously been linked to its degradation following UV exposure, this is the first report linking a HAT-mediated PCNA acetylation to PCNA monoubiquitination. These findings add a new dimension to our knowledge of the mechanisms regulating PCNA ubiquitination post-UV exposure in eukaryotes.

Characterization of the MCM homohexamer from the thermoacidophilic euryarchaeon Picrophilus torridus.

The typical archaeal MCM exhibits helicase activity independently in vitro. This study characterizes MCM from the euryarchaeon Picrophilus torridus. While PtMCM hydrolyzes ATP in DNA-independent manner, it displays very poor ability to unwind DNA independently, and then too only under acidic conditions. The protein exists stably in complex with PtGINS in whole cell lysates, interacting directly with PtGINS under neutral and acidic conditions. GINS strongly activates MCM helicase activity, but only at low pH. In consonance with this, PtGINS activates PtMCM-mediated ATP hydrolysis only at low pH, with the amount of ATP hydrolyzed during the helicase reaction increasing more than fifty-fold in the presence of GINS. While the stimulation of MCM-mediated helicase activity by GINS has been reported in MCMs from P.furiosus, T.kodakarensis, and very recently, T.acidophilum, to the best of our knowledge, this is the first report of an MCM helicase demonstrating DNA unwinding activity only at such acidic pH, across all archaea and eukaryotes. PtGINS may induce/stabilize a conducive conformation of PtMCM under acidic conditions, favouring PtMCM-mediated DNA unwinding coupled to ATP hydrolysis. Our findings underscore the existence of divergent modes of replication regulation among archaea and the importance of investigating replication events in more archaeal organisms.

Characterization of the replication initiator Orc1/Cdc6 from the Archaeon Picrophilus torridus.

Eukaryotic DNA replication is preceded by the assembly of prereplication complexes (pre-RCs) at or very near origins in G1 phase, which licenses origin firing in S phase. The archaeal DNA replication machinery broadly resembles the eukaryal apparatus, though simpler in form. The eukaryotic replication initiator origin recognition complex (ORC), which serially recruits Cdc6 and other pre-RC proteins, comprises six components, Orc1-6. In archaea, a single gene encodes a protein similar to both the eukaryotic Cdc6 and the Orc1 subunit of the eukaryotic ORC, with most archaea possessing one to three Orc1/Cdc6 orthologs. Genome sequence analysis of the extreme acidophile Picrophilus torridus revealed a single Orc1/Cdc6 (PtOrc1/Cdc6). Biochemical analyses show MBP-tagged PtOrc1/Cdc6 to preferentially bind ORB (origin recognition box) sequences. The protein hydrolyzes ATP in a DNA-independent manner, though DNA inhibits MBP-PtOrc1/Cdc6-mediated ATP hydrolysis. PtOrc1/Cdc6 exists in stable complex with PCNA in Picrophilus extracts, and MBP-PtOrc1/Cdc6 interacts directly with PCNA through a PIP box near its C terminus. Furthermore, PCNA stimulates MBP-PtOrc1/Cdc6-mediated ATP hydrolysis in a DNA-dependent manner. This is the first study reporting a direct interaction between Orc1/Cdc6 and PCNA in archaea. The bacterial initiator DnaA is converted from an active to an inactive form by ATP hydrolysis, a process greatly facilitated by the bacterial ortholog of PCNA, the ß subunit of Pol III. The stimulation of PtOrc1/Cdc6-mediated ATP hydrolysis by PCNA and the conservation of PCNA-interacting protein motifs in several archaeal PCNAs suggest the possibility of a similar mechanism of regulation existing in archaea. This mechanism may involve other yet to be identified archaeal proteins.

A highly basic sequence at the N-terminal region is essential for targeting the DNA replication protein ORC1 to the nucleus in Leishmania donovani.

The conserved eukaryotic DNA replication protein ORC1 is one of the constituents of pre-replication complexes that assemble at or very near origins prior to replication initiation. ORC1 has been shown to be constitutively nuclear in Leishmania major. This study investigates the sequences involved in nuclear localization of ORC1 in Leishmania donovani, the causative agent of visceral leishmaniasis. Nuclear localization signals (NLSs) have been reported in only a few Leishmania proteins. Functional analyses have delineated NLSs to regions of ~60 amino acids in length in the tyrosyl DNA phosphodiesterase I and type II DNA topoisomerase of L. donovani, and in the L. major kinesin KIN13-1. Using a panel of site-directed mutations we have identified a sequence essential for nuclear import of LdORC1. This sequence at the N terminus of the protein comprises residues 2-5 (KRSR), with K2, R3 and R5 being crucial. Independent mutation of the K2 residue causes exclusion of the protein from the nucleus, while mutating the R5 residue leads to diffusion of the protein throughout the cell. This sequence, however, is insufficient for targeting a heterologous protein (ß-galactosidase) to the nucleus. Analysis of additional ORC1 mutations and reporter constructs reveals that while the highly basic tetra-amino acid sequence at the N terminus is essential for nuclear localization, the ORC1 NLS in its entirety is more complex, and of a distributive character. Our results suggest that nuclear localization signalling sequences in Leishmania nuclear proteins are more complex than what is typically seen in higher eukaryotes.