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Organic molecules that exhibit charge-transfer (CT) excited states are known to play an important role in processes linked to electron transfer properties and molecular conductance. In this article, we present a simple technique based on "Becke's excitation theorem" that offers an accurate picture of these electronic states. It expresses the correlated energy splitting between triplet and its corresponding singlet states by a two-electron integral, which is numerically evaluated by our recently developed strategy on Cartesian grid. We first examine the consistency of our adopted numerical strategy to evaluate the integral with the original prescribed technique. Then we assess the method on weakly bound CT complexes with three different functionals (BLYP, B3LYP, and LC-BLYP). The accuracy on asymptotic limit of CT excitation is also explored. Finally in order to illustrate the strength and feasibility, it is further extended to a few "challenging" molecules. The method, when employed with hybrid B3LYP functional, turns out to be quite accurate to describe CT excitation energy.

Photoluminescence effects in organic chromophores are of significant importance and requires precise description of low lying excited states. In this communication, we put forward an alternative time-independent DFT scheme for computing lowest single-particle excitation energy, especially for singlet excited state. This adopts a recently developed "virial"-theorem based model of singlet-triplet splitting which requires a DFT calculation on closed shell ground state and a restricted open-shell triplet excited state, followed by a simple 2e- integral evaluation. This produces vertical excitation energies in small molecules, linear and non-linear polycyclic aromatic hydrocarbon and organic dyes in comparable accuracy to the TDDFT. We also explore the functional dependency of present method with three different functionals (B3LYP, wB97X and CAM-B3LYP) for polyenes and linear acenes. A systematic comparison with literature value illustrates the validity and usefulness of the present scheme in determining optical gap with fair computational cost.

Circulating tumor DNA (ctDNA) sequencing is being rapidly adopted in precision oncology, but the accuracy, sensitivity, and reproducibility of ctDNA assays is poorly understood. Here we report the findings of a multi-site, cross-platform evaluation of the analytical performance of five industry-leading ctDNA assays. We evaluated each stage of the ctDNA sequencing workflow with simulations, synthetic DNA spike-in experiments, and proficiency testing on standardized cell line–derived reference samples. Above 0.5% variant allele frequency, ctDNA mutations were detected with high sensitivity, precision and reproducibility by all five assays, whereas below this limit detection became unreliable and varied widely between assays, especially when input material was limited. Missed mutations (false-negatives) were more common than erroneous candidates (false-positives), indicating that the reliable sampling of rare ctDNA fragments is the key challenge for ctDNA assays. This comprehensive evaluation of the analytical performance of ctDNA assays serves to inform best-practice guidelines and provides a resource for precision oncology., Editorial summary: Reliable detection of mutations below 0.5% variant allele frequency remains a key challenge for circulating tumor DNA sequencing assays.

Postcolonial discourses often view globality as marking the continuation of the imperialist project. However, discourses entailing a genetic assessment of globality have identified that the workings of the neoliberal economy are largely responsible for its undoing. This mutually destructive relationship between globality and neoliberalism makes it even more necessary to strike a rupture between them. This article illustrates the strands of global and neoliberal discontent, positioning both globality and neoliberalism as arriving at cul-de sac despite vigorous effort to pretend otherwise. In particular, it dwells on the ontological status of the migrants in India by discussing the current strategy to criminalize them and uses Rahul Pandita’s Our Moon has Blood Clots: A Memoir of a Lost Home in Kashmir to show how criminalized migrants may be turned, by de-globing, into natural inhabitants of the Earth.

This paper is an analysis of the plasticity of love and its appropriation as an effective strategy for those in power to deal with the refugee crisis. It contends that a contextualization of love is needed to point to some of the ways in which love’s plasticity is employed to combat refugee uprisings. Through an analysis of Mohsin Hamid’s Exit West (2017) and Sharon Bala’s The Boat People (2018), this paper demonstrates how the ruling governments, of different contexts, either overtly or covertly, resort to politicizing love in order to defuse tensions between refugees and “insiders”.

In this communication, we represent a self-consistent systematic optimization procedure for the development of optimally tuned (OT) range-separated hybrid (RSH) functionals from \emph{first principles}. This is an offshoot of our recent work, which employed a purely numerical approach for efficient computation of exact exchange contribution in the conventional global hybrid functionals through a range-separated (RS) technique. We make use of the size-dependency based ansatz i.e., RS parameter, $\gamma$, is a functional of density, $\rho(\mathbf{r})$, of which not much is known. To be consistent with this ansatz, a novel procedure is presented that relates the characteristic length of a given system (where $\rho(\mathbf{r})$ exponentially decays to zero) with $\gamma$ self-consistently via a simple mathematical constraint. In practice, $\gamma_{\mathrm{OT}}$ is obtained through an optimization of total energy as follows: $\gamma_{\mathrm{OT}} \equiv \underset{\gamma }{\mathrm{opt}} \ E_{\mathrm{tot},\gamma}$. It is found that the parameter $\gamma_{\mathrm{OT}}$, estimated as above can show better performance in predicting properties (especially from frontier orbital energies) than conventional respective RSH functionals, of a given system. We have examined the nature of highest fractionally occupied orbital from exact piece-wise linearity behavior, which reveals that this approach is sufficient to maintain this condition. A careful statistical analysis then illustrates the viability and suitability of the current approach. All the calculations are done in a Cartesian-grid based pseudopotential (G)KS-DFT framework., Comment: 34 pages, 8 tables, 4 figures

Photoluminescence effects in organic chromophores are of significant importance and requires precise description of low lying excited states. In this communication, we put forward an alternative time-independent DFT scheme for computing lowest single-particle excitation energy, especially for singlet excited state. This adopts a recently developed "virial"-theorem based model of singlet-triplet splitting which requires a DFT calculation on closed shell ground state and a restricted open-shell triplet excited state, followed by a simple

Photon-induced electronic excitations are ubiquitously observed in organic chromophore. In this context, we present a simple, alternative time-independent DFT procedure, for the computation of single-particle excitation energies, in particular, the lower bound excited singlet states, which are of primary interest in photochemistry. This takes inspiration from recently developed Becke’s exciton model, where a key step constitutes the accurate evaluation of correlated singlet–triplet splitting energy. It introduces a non-empirical model, from both “adiabatic connection theorem” and “virial theorem” to analyze the role of 2e $$^-$$ integral in such calculations. The latter quantity is efficiently mapped onto a real grid and computed accurately using a purely numerical strategy. Illustrative calculations are performed on 10 $$\pi $$ -electron organic chromophores within a Cartesian grid implementation of pseudopotential Kohn–Sham (KS) DFT, developed in our laboratory, taking SBKJC-type basis functions within B3LYP approximation. The triplet and singlet excitation energies corresponding to first singly excited configuration are found to be in excellent agreement with TD-B3LYP calculations. Further, we perform the same for a set of larger molecular systems using the asymptotically corrected LC-BLYP, in addition to B3LYP. A systematic comparison with theoretical best estimates demonstrates the viability and suitability of current approach in determining optical gaps, combining predictive accuracy with moderate computational cost.

Background Circulating tumor (ct) DNA assays performed in clinical laboratories provide tumor biomarker testing support for biopharmaceutical clinical trials. Yet it is neither practical nor economically feasible for many of these clinical laboratories to internally develop their own liquid biopsy assay. Commercially available ctDNA kits are a potential solution for laboratories seeking to incorporate liquid biopsy into their test menus. However, the scarcity of characterized patient samples and cost of purchasing validation reference standards creates a barrier to entry. In the current study, we evaluated the analytical performance of the AVENIO ctDNA liquid biopsy platform (Roche Sequencing Solutions) for use in our clinical laboratory. Method Intra-laboratory performance evaluation of AVENIO ctDNA Targeted, Expanded, and Surveillance kits (Research Use Only) was performed according to College of American Pathologists (CAP) guidelines for the validation of targeted next generation sequencing assays using purchased reference standards, de-identified human plasma cell-free (cf) DNA samples, and contrived samples derived from commercially purchased normal and cancer human plasma. All samples were sequenced at read depths relevant to clinical settings using the NextSeq High Output kit (Illumina). Results At the clinically relevant read depth, Avenio ctDNA kits demonstrated 100% sensitivity in detecting single nucleotide variants (SNVs) at ≥0.5% allele frequency (AF) and 50% sensitivity in detecting SNVs at 0.1% AF using 20–40 ng sample input amount. The assay integrated seamlessly into our laboratory’s NGS workflow with input DNA mass, target allele frequency (TAF), multiplexing, and number of reads optimized to support a high-throughput assay appropriate for biopharmaceutical trials. Conclusions Our study demonstrates that AVENIO ctDNA liquid biopsy platform provides a viable alternative for efficient incorporation of liquid biopsy assays into the clinical laboratory for detecting somatic alterations as low as 0.5%. Accurate detection of variants lower than 0.5% could potentially be achieved by deeper sequencing when clinically indicated and economically feasible.

Additional file 5: Supplementary Table S2. AVENIO Expanded ctDNA analysis kit sensitivity (SNV/INDEL)

Additional file 6: Supplementary Table S3. Avenio Surveillance ctDNA kit sensitivity (SNV/INDEL)

Additional file 8: Supplementary Table S5. INDEL Loci of interest for Avenio Expanded kit

Additional file 9: Supplementary Table S6. INDEL Loci of interest for Avenio Targeted and Surveillance kit

Additional file 7: Supplementary Table S4. Avenio Targeted ctDNA kit sensitivity (SNV/INDEL)

Additional file 4: Supplementary Table S1. Sample QC Metrics across three panels [Targeted, Expanded, Surveillance]

Background: Paired-box Pax gene family protein 5 (PAX5)/B-cell specific activator protein (BSAP) is a transcription factor encoded by the PAX5 gene and has an essential role in B-cell differentiation and maturation. High PAX5 expression is detected ensures commitment to B-cell lineage. PAX5 is normally downregulated at the plasma cell stage of B-cell development. Complete or partial deletion of the PAX5 gene has been found as secondary event associated with BCR-ABL1 or TCF3-PBX1 fusions in Acute Lymphocytic Leukemia (ALL) cases. PAX5 expression is a diagnostic marker for B-cell lineage and may help quantify minimal residual disease in B-ALL. Lineage determination of leukemic blasts is most often performed by flow cytometry, but also by immunohistochemistry (IHC). Evaluation of PAX5 is most commonly available by IHC and is not widely performed by flow cytometry. In cases with limited specimen for evaluation or aberrant loss of some B-cell markers, determining quantitative levels of RNA from lineage-specific genes, such as PAX5, could be a valuable clinical diagnostic tool for ALL patients. Our existing single tube NGS based assay for simultaneous detection of DNA alterations and RNA fusions in heme malignancies from Total Nucleic Acid (TNA), can also be used to detect PAX5 gene expression through select exons enrichment along with a total of 213 genes. However, one of the current challenges for NGS-based gene expression profiling is to setup a threshold for overexpression. Here we developed a cutoff criterion for PAX5 overexpression and evaluated the performance of PAX5 gene expression analysis using the in-use heme assay and its potential use in clinical laboratory for cell lineage detection. Methods: RNA sequencing was performed on TNA extracted from ALL samples and from 32 healthy donors using partial anchored amplicon based (Qiagen, inc) heme NGS assay. PAX5 RNA expression was calculated by TPM (transcript per million) counts normalized to TPM of the house-keeping gene GUSB. A commercially available qRT-PCR assay was used as orthogonal method to confirm the gene expression. The expression call by NGS based on the normalized value was confirmed by a commercial qRT-PCR assay in house validated through serial dilutions of template for six log scale. The analytical cutoff was determined from normalized TPM calculation from 32 healthy volunteers following CLSI guideline (CLSI_EP17-A2) and evaluated the outcome with IHC positive /negative clinical samples (a CLIA validated assay). Further, we used the established cutoff to evaluate the sensitivity or specificity in cohort of ALL samples. Results: In this study we established the cutoff for PAX5 gene over-expression using the currently in-use heme NGS assay. First, a cutoff was established following the method in the CLSI guidelines and tested for sensitivity and specificity in the ALL sample cohort. PAX5 TPM normalization to GUSB or to the geometric mean of four house keeping genes (GUSB, PGD, RPL5 and RPL19) showed a strong correlation (R2>0.95), and GUSB was selected for further normalization since GUSB TPM values were most conserved across all the samples. Independent in-house evaluation for commercial qRT-PCR assay showed efficiency at 94.3 and 96% for GUSB and PAX5, respectively (with linearity R2>0.95), and been used to compare the NGS and IHC data as independent orthogonal assay. When a cohort of samples for Pax5 by IHC (positive and negative), a sensitivity at 67% and specificity at 100% were observed for the NGS based Pax5 detection. NGS results on the discordant samples were confirmed by qRT-PCR to have low RNA expression. Notably the discordant, IHC positive samples contained very low numbers of B cells. Alongside with other possible mechanisms of increased protein levels such as increased protein translation/increased protein stability could explain the discordance between RNA expression and the protein detection by IHC. Conclusions: In this study we demonstrate that NeoGenomics's (heme) NGS assay can be used for PAX5 gene over-expression analysis on ALL. The heme NGS is inexpensive and is already integrated in the benchwork workflow without adding extra burden and can be used as an objective quantification of PAX5 levels overcoming the challenges associated with the relative signal intensity biases in IHC testing. This type of RNA testing can be useful especially with specimens having limited material. Disclosures Ghosal: NeoGenomics Laboratories: Current Employment. Alarcon: NeoGenomics Laboratories: Current Employment. Koo: Neo Genomics Laboratories: Current Employment. Kang: Neo Genomics Laboratories: Current Employment. Ramesh: Neo Genomics Laboratories: Current Employment. Gyuris: Neo Genomics Laboratories: Current Employment. Jung: NeoGenomics Laboratories, Inc.: Current Employment. Thomas: NeoGenomics Laboratories, Inc.: Current Employment. Fabunan: NeoGenomics Laboratories, Inc.: Current Employment. Magnan: NeoGenomics Laboratories, Inc.: Current Employment. Nam: NeoGenomics Laboratories, Inc.: Current Employment. Petersen: Neo Genomics Laboratories: Current Employment. Lopez-Diaz: NeoGenomics Laboratories, Inc.: Current Employment. Bender: NeoGenomics Laboratories, Inc.: Current Employment. Agersborg: NeoGenomics Laboratories, Inc.: Current Employment. Ye: Neo Genomics Laboratories: Current Employment. Funari: NeoGenomics Laboratories, Inc.: Current Employment.

Background: Acute Lymphocytic Leukemia (ALL) is the most common childhood cancer and accounts for about a quarter of adult acute leukemias. Current NCCN recommendations for clinical testing for risk stratification and treatment guidance include karyotyping, FISH testing for translocations, and RT-PCR for gene fusions and sequencing for DNA mutations detection. Most NGS based approaches test DNA mutations and RNA fusions separately, thereby requiring higher input material and multiple workflows adding to the cost and turn-around-time. An NGS based assay for the detection of DNA variants (NeoGenomics Heme NGS assay) in heme malignancies using Total Nucleic Acid (TNA) is already available in our clinical laboratory and complements FISH based fusion detection and karyotyping but an integral assay to detect both DNA and RNA alterations with a simple workflow for ALL is needed. Methods: We used TNA or RNA spiked-in with DNA to simulate TNA samples, extracted from 93 bone marrow and peripheral blood samples from patients and healthy donors, along with commercial fusion reference myeloid samples Seraseq Myeloid Fusion RNA Mix (SeraCare Inc.) controls. DNA/RNA libraries were prepared using a custom amplicon based Multimodal NGS panel (Qiagen Inc.) targeting 297 genes and 213 genes (select exons) for DNA and RNA fusion detection, respectively. The enriched dual indexed amplicon libraries were sequenced on an Illumina NovaSeq 6000. The sequence data was processed with a customized bioinformatic pipeline for DNA variant as well as a novel machine learning algorithm for RNA fusion detection. We analyzed sensitivity, specificity, accuracy, reproducibility, and repeatability for clinical use. The DNA variants were orthogonally confirmed using other NGS assays, and the RNA fusions were confirmed on an RNA-seq Archer assay or RT-Sanger confirmation assays. Results: Here, we developed and validated a single tube comprehensive NGS panel using a custom multimodal chemistry that uses TNA as input for simultaneous dual detection of DNA and RNA abnormalities in ALL patients' samples. We performed the analytical validation of our Heme NGS assay for the RNA panel to detect fusions in ALL, using TNA input for comprehensive DNA and RNA mutation detection. The fusion concordance was 95% for the RNA fusion panel. The assay detected BCR-ABL1 (7/7), ETV6-RUNX1 (1/1), KMT2A fusions (4/5), TCF3-PBX1 (1/1), and PCM1-JAK2(1/1). The specificity was determined at 100% using a set of 42 fusion negative samples. The limit of detection (LOD) was analyzed using serial dilutions to up to 3 log reduction (LR) using a the Seraseq Myeloid Fusion sample. The fusions were detected down to 1 LR. The reproducibility was tested using a positive fusion and Seraseq samples across three runs and was reported at 100%. Next, a small cohort of ALL samples (n=8) was included as part of this study to simultaneously evaluate DNA and RNA mutations. We detected pathogenic DNA variants in genes previously reported in ALL that included NOTCH1, PTEN, FLT3, IKZF1, JAK1, JAK2, KRAS, NF1, PAX5, U2AF1, TP53, and also RNA fusion BCR-ABL1, and the results were confirmed by an orthogonal NGS assay (NexCourse and RNA-Seqv1 for fusions). One sample carrying a BCR-ABL1 fusion (detected by RNA panel) also harbored mutations in IKZF1 in DNA (detected by DNA panel) that is reported as unfavorable prognostic biomarker for Ph-Like ALL demonstrating comprehensive panel could identify multiple variants within the same sample, demonstrating the advantage DNA+RNA testing has over the classical single gene FISH/RT-PCR testing for the efficient risk stratification and treatment in ALL patients. Conclusions: In this study, we demonstrated that the single tube TNA based NeoGenomics NGS assay can simultaneously detect the DNA and RNA biomarkers associated with ALL for improved diagnostic and prognostic recommendations. The single-tube assay for detection of both RNA fusions and DNA variants using the same sample could offer comprehensive and cost-effective solution for clinical laboratory test for ALL patient care. This is a promising approach that might be used as a dual DNA/RNA alterations detection on other hematological neoplasia. Disclosures Ramesh: Neo Genomics Laboratories: Current Employment. Koo: Neo Genomics Laboratories: Current Employment. Kang: Neo Genomics Laboratories: Current Employment. Ghosal: NeoGenomics Laboratories: Current Employment. Alarcon: NeoGenomics Laboratories: Current Employment. Gyuris: Neo Genomics Laboratories: Current Employment. Jung: NeoGenomics Laboratories, Inc.: Current Employment. Magnan: NeoGenomics Laboratories, Inc.: Current Employment. Nam: NeoGenomics Laboratories, Inc.: Current Employment. Thomas: NeoGenomics Laboratories, Inc.: Current Employment. Fabunan: NeoGenomics Laboratories, Inc.: Current Employment. Petersen: Neo Genomics Laboratories: Current Employment. Lopez-Diaz: NeoGenomics Laboratories, Inc.: Current Employment. Bender: NeoGenomics Laboratories, Inc.: Current Employment. Agersborg: NeoGenomics Laboratories, Inc.: Current Employment. Ye: Neo Genomics Laboratories: Current Employment. Funari: NeoGenomics Laboratories, Inc.: Current Employment.

Background: Multiple myeloma (MM) is a blood cancer type affecting plasma cell in bone marrow. MM is heterogenous in nature but t(11;14)(q13;q32) translocation is a common prognostic marker among MM patients. One of the most frequent oncogenic drivers involved in this chromosomal rearrangement is CCND1 (Cyclin D1) gene translocation downstream to the immunoglobulin heavy chain (IGH), which results on overexpression of CCND1, thus promoting abnormal cell proliferation. Oncogenic CCND1 RNA levels can result from translocations such as t(11;14), gene amplifications, increased transcription rates and/or RNA stability. Indeed, CCND1 RNA overexpression has a favorable prognostic value for patients treated with high doses of chemotherapies but important challenges remain in accurate detection of CCND1 RNA levels. Currently, FISH is the gold standard method for detecting t(11:14) translocations at the DNA level. However, it cannot detect CCND1 overexpression. Therefore, a method that can detect CCND1 overexpression levels, as well as in frame transcripts has clinical implications. In the current study we leveraged in-use NeoGenomics Heme TNA single tube NGS assay to enable the detection of CCND1 RNA overexpression as a complementary test to FISH testing. Methods: We performed RNA sequencing from 32 healthy donors and on fixed cell pellets from 94 CD138-enriched BM samples from MM patients and from using the amplicon based (Qiagen, inc) NGS assay. We developed pipeline for gene expression by TPM count (transcript per million) for CCND1, and further normalized to the "housekeeping" gene GUSB. We validated the normalization to GUSB by comparing to normalization using the geometric mean of four housekeeping genes (GUSB, PGD, RPL5 and RPL19) showing a high correlation (R 2>0.95). A commercially available qRT-PCR assay was used as orthogonal method to further confirm the linearity of the quantitative gene expression signal in NGS. The analytical cutoff was determined from normalized TPM calculation from 32 healthy volunteers following CLSI guideline (CLSI_EP17-A2) and further updated from MM-PCE samples with t(11:14) translocations from a CLIS-validated FISH assay . Results: From 94 CD138-enriched BM samples, 26 had t(11:14) translocations, or CCND1 gains as detected by FISH, 15 samples were confirmed negative by FISH and 32 normal volunteers with no suspected disease. Also, we determined the analytical cutoff for CCND1 overexpression based on the CLSI guidelines to be 2.37 times the expression level of GUSB ("housekeeping" gene) using normal volunteers (n=32) (sensitivity 86% and specificity 77%). We found specificity to be low, so further evaluated the threshold using a ROC curve analysis with multiple tests. Using Fischer's exact test, we found CCND1 expression 3.27 times the GUSB expression to yield higher specificity of 86.5 % and sensitivity for 78.9%. Further, we used 26 FISH positive and 32 normal samples to build a new model and determined the cutoff for CCND1 overexpression to be 4.15 times GUSB expression, which resulted lower sensitivity but higher specificity (75% sensitivity and 100% specificity). When we evaluated 15 FISH negative samples with this cutoff we observed CCND1 was not overexpressed in six samples, but 9 samples did have some degree of overexpression. Overexpression was confirmed by qRT-PCR. Two CCND1 high- and low- expressing normal samples (MM-PCE 27 and 48) were further evaluated using alternative extraction methods to test the dependencies on extractions and the data showed concordant to each other for overexpression. Interestingly, 1 sample (MM-PCE-27) showed very high overexpression without t(11:14) translocation event (~100 fold over expressed). Cytogenetic studies were discordant with FISH as well for this sample, showing abnormalities related to chr7q,13q,12p but no indication of any chr11 related event. Conclusions: In this study we evaluated our existing NGS assay for CCND1 overexpression using TNA as a surrogate for traditional FISH, while demonstrating the accuracy of the RNA quantitation by NGS using qRT-PCR. We developed an RNA-seq based CCND1 expression assay that could be used to complement traditional FISH testing especially if there is limited specimen. The confirmation of overexpression in FISH negative samples may suggest new ways to improve MM patients risk stratification and treatment. Disclosures Ghosal: NeoGenomics Laboratories: Current Employment. Alarcon: NeoGenomics Laboratories: Current Employment. Koo: Neo Genomics Laboratories: Current Employment. Kang: Neo Genomics Laboratories: Current Employment. Ramesh: Neo Genomics Laboratories: Current Employment. Gyuris: Neo Genomics Laboratories: Current Employment. Jung: NeoGenomics Laboratories, Inc.: Current Employment. Thomas: NeoGenomics Laboratories, Inc.: Current Employment. Fabunan: NeoGenomics Laboratories, Inc.: Current Employment. Magnan: NeoGenomics Laboratories, Inc.: Current Employment. Nam: NeoGenomics Laboratories, Inc.: Current Employment. Petersen: Neo Genomics Laboratories: Current Employment. Lopez-Diaz: NeoGenomics Laboratories, Inc.: Current Employment. Yamahata: Neo Genomics Laboratories: Current Employment. Bender: NeoGenomics Laboratories, Inc.: Current Employment. Agersborg: NeoGenomics Laboratories, Inc.: Current Employment. Ye: Neo Genomics Laboratories: Current Employment. Funari: NeoGenomics Laboratories, Inc.: Current Employment.

Static electric response properties of atoms and molecules are reported within the real-space Cartesian grid implementation of pseudopotential Kohn-Sham (KS) density functional theory (DFT). A detailed systematic investigation is made for a representative set of atoms and molecules, through a number of properties like total ground-state electronic energies, permanent dipole moment ($\bm{\mu}$), static average dipole polarizability ($\overline{\alpha}$). This is further extended to first-hyperpolarizability ($\bm{\beta}$) in molecules. It employs a recently developed non-uniform grid-optimization technique, with a suitably chosen fixed initial applied field. A simple variant of the finite-field method, using a rational function fit to the dipole moment with respect to electric field, is adopted. We make use of Labello-Ferreira-Kurtz (LFK) basis set, which has performed quite well in these scenarios. To assess the efficacy and feasibility, four XC functionals such as LDA, BLYP, PBE and LBVWN are chosen. Present results are compared with available literature (both theoretical and experimental) values, whenever possible. In all instances, they show excellent agreement with the respective atom-centered-grid results, very widely used in many quantum chemistry programs. This demonstrates a viable alternative towards accurate prediction of response properties of many-electron systems in Cartesian coordinate grid., Comment: 18 pages, 4 tables

Within the finite-field Kohn-Sham framework, static electric response properties of diatomic molecules are presented. The electronic energy, dipole moment ({\boldmath$\mu$}), static dipole polarizability ({\boldmath$\alpha$}) and first-hyperpolarizability ({\boldmath$\beta$}) are calculated through a pseudopotential-DFT implementation in Cartesian coordinate grid, developed in our laboratory earlier. We engage the Labello-Ferreira-Kurtz (LFK) basis set; while four local and non-local exchange-correlation (LDA, BLYP, PBE and LBVWN) functionals have been adopted. A detailed analysis of \emph{grid convergence} and its impact on obtained results, is presented. In each case the \emph{electric field optimization} was carefully monitored through a recently prescribed technique. For all three molecules (HCl, HBr, HI) considered, the agreement of all these quantities with widely successful and popular atom-centered-grid procedure, is excellent. To assess the efficacy and feasibility, companion calculations are performed for these on a representative molecule (HCl) at distorted geometries, far from equilibrium. Wherever possible, relevant comparison is made with available \emph{all-electron} data and experimental results. This demonstrates that Cartesian grid provides accurate, reliable results for such properties of many-electron systems within pseudopotential representation., Comment: 25 pages, 4 tables, 2 figures

Information-based uncertainty measures like Shannon entropy, Onicescu energy and Fisher information (in position and momentum space) are employed to understand the effect of \emph{symmetric and asymmetric} confinement in a quantum harmonic oscillator. Also, the transformation of Hamiltonian into a dimensionless form gives an idea of the composite effect of force constant and confinement length ($x_c$). In symmetric case, a wide range of $x_{c}$ has been taken up, whereas asymmetric confinement is dealt by shifting the minimum of potential from origin keeping box length and boundary fixed. Eigenvalues and eigenvectors for these systems are obtained quite accurately via an imaginary time propagation scheme. For asymmetric confinement, a variation-induced exact diagonalization procedure is also introduced, which produces very high-quality results. One finds that, in symmetric confinement, after a certain characteristic $x_{c}$, all these properties converge to respective values of free harmonic oscillator. In asymmetric situation, excited-state energies always pass through a maximum. For this potential, the classical turning-point decreases, whereas well depth increases with the strength of asymmetry. Study of these uncertainty measures reveals that, localization increases with an increase of asymmetric parameter.

The widely distributed colonization factor (CF) CS6 of enterotoxigenic Escherichia coli (ETEC) has gained importance over the years in terms of its structure and function. CS6 is an afimbrial assembly in contrast to the other ETEC CFs, which are mostly fimbrial. A recent study predicted a linear fibre model for recombinant chimeric CS6 and formation of oligomers in solution. In this study, we characterized the oligomeric assembly of CS6, purified from a clinical ETEC isolate and identified its existence in the WT strain. We found that purified CS6 forms a continuous array of higher order oligomers composed of two tightly associated subunits, CssA and CssB in an equal (1:1) stoichiometry. This oligomerization occurs by formation of (CssA-CssB)n complex where 'n' increases with the concentration. The diameter of CS6 oligomers also proportionally increases with concentration. More significantly, we showed CS6 oligomers to be spherical in shape instead of being linear fibres as predicted earlier and this was further confirmed by electron microscopy. We also showed CS6 assembled on the bacterial surface in the form of an oligomeric complex. This process depends on the expression of properly folded CssA and CssB together, guided by the chaperone CssC and usher CssD. In conclusion, our results provide evidence for the existence of concentration-dependent, spherical oligomers of CS6 comprising both the structural subunits in equal stoichiometry and the CS6 oligomeric complex on the ETEC surface.

Infection with the nontyphoidal Salmonella is a common cause of food-borne disease that leads to acute gastroenteritis/diarrhea. Severe/prolonged cases of Salmonella infection could also impact host nutritional status, but little is known about its effect on intestinal absorption of vitamins, including biotin. We examined the effect of Salmonella enterica serovar Typhimurium ( S. typhimurium) infection on intestinal biotin uptake using in vivo (streptomycin-pretreated mice) and in vitro [mouse (YAMC) and human (NCM460) colonic epithelial cells, and human intestinal epithelial Caco-2 cells] models. The results showed that infecting mice with wild-type S. typhimurium, but not with its nonpathogenic isogenic invA spiB mutant, leads to a significant inhibition in jejunal/colonic biotin uptake and in level of expression of the biotin transporter, sodium-dependent multivitamin transporter. In contrast, infecting YAMC, NCM460, and Caco-2 cells with S. typhimurium did not affect biotin uptake. These findings suggest that the effect of S. typhimurium infection is indirect and is likely mediated by proinflammatory cytokines, the levels of which were markedly induced in the intestine of S. typhimurium-infected mice. Consistent with this hypothesis, exposure of NCM460 cells to the proinflammatory cytokines TNF-α and IFN-γ led to a significant inhibition of biotin uptake, sodium-dependent multivitamin transporter expression, and activity of the SLC5A6 promoter. The latter effects appear to be mediated, at least in part, via the NF-κB signaling pathway. These results demonstrate that S. typhimurium infection inhibits intestinal biotin uptake, and that the inhibition is mediated via the action of proinflammatory cytokines.

We present a purely numerical approach in Cartesian grid, for efficient computation of Hartree-Fock (HF) exchange contribution in the HF and density functional theory models. This takes inspiration from a recently developed algorithm [Liu \emph{et al.}, J.~Chem.~Theor.~Comput.~ \textbf{13}, 2571 (2017)]. A key component is the accurate evaluation of electrostatic potential integral, which is the rate-determining step. This introduces the Fourier convolution theorem in conjunction with a range-separated Coulomb interaction kernel. The latter is efficiently mapped into real grid through a simple optimization procedure, giving rise to a constraint in the range-separated parameter. The overall process offers logarithmic scaling with respect to molecular size. It is then extended towards global hybrid functionals such as B3LYP, PBE0 and BHLYP within pseudopotential Kohn-Sham theory, through an LCAO-MO ansatz in Cartesian grid, developed earlier in our laboratory. For sake of comparison, a parallel semi-numerical approach has also been worked out that exploits the familiar Obara-Saika recursion algorithm. An excellent agreement between these two routes is demonstrated through total energy and orbital energy in a series of atoms and molecules (including 10 $\pi$-electron molecules), employing an LANL2DZ-type basis function. A critical analysis of these two algorithms reveals that the proposed numerical scheme could lead to very attractive and competitive scaling. The success of our approach also enables us for further development of optimally tuned range-separated hybrid and hyper functionals., Comment: 26 pages, 6 tables, 66 references

Intestinal epithelial cells undergo differentiation as they move from the crypt to the villi, a process that is associated with up- and downregulation in expression of a variety of genes, including those involved in nutrient absorption. Whether the intestinal uptake process of vitamin B2[riboflavin (RF)] also undergoes differentiation-dependent regulation and the mechanism through which this occurs are not known. We used human-derived intestinal epithelial Caco-2 cells and native rat intestine as models to address these issues. Caco-2 cells showed a significantly higher carrier-mediated RF uptake in post- than preconfluent cells. This upregulation was associated with a significantly higher level of protein and mRNA expression of the RF transporters hRFVT-1 and hRFVT-3 in the post- than preconfluent cells; it was also accompanied with a significantly higher rate of transcription of the respective genes ( SLC52A1 and SLC52A3), as indicated by the higher level of expression of heterogeneous nuclear RNA and higher promoter activity in post- than preconfluent cells. Studies with native rat intestine also showed a significantly higher RF uptake by epithelial cells of the villus tip than epithelial cells of the crypt; this again was accompanied by a significantly higher level of expression of the rat RFVT-1 and RFVT-3 at the protein, mRNA, and heterogeneous nuclear RNA levels. These findings show, for the first time, that the intestinal RF uptake process undergoes differentiation-dependent upregulation and suggest that this is mediated (at least in part) via transcriptional mechanisms.

The Slc5a6 gene expresses a plasma membrane protein involved in the transport of the water-soluble vitamin biotin; the transporter is commonly referred to as the sodium-dependent multivitamin transporter (SMVT) because it also transports pantothenic acid and lipoic acid. The relative contribution of the SMVT system toward carrier-mediated biotin uptake in the native intestine in vivo has not been established. We used a Cre/lox technology to generate an intestine-specific (conditional) SMVT knockout (KO) mouse model to address this issue. The KO mice exhibited absence of expression of SMVT in the intestine compared with sex-matched littermates as well as the expected normal SMVT expression in other tissues. About two-thirds of the KO mice died prematurely between the age of 6 and 10 wk. Growth retardation, decreased bone density, decreased bone length, and decreased biotin status were observed in the KO mice. Microscopic analysis showed histological abnormalities in the small bowel (shortened villi, dysplasia) and cecum (chronic active inflammation, dysplasia) of the KO mice. In vivo (and in vitro) transport studies showed complete inhibition in carrier-mediated biotin uptake in the intestine of the KO mice compared with their control littermates. These studies provide the first in vivo confirmation in native intestine that SMVT is solely responsible for intestinal biotin uptake. These studies also provide evidence for a casual association between SMVT function and normal intestinal health.

Utilizing a conditional (intestinal-specific) knockout (cKO) mouse model, we have recently shown that the sodium-dependent multivitamin transporter (SMVT) ( SLC5A6) is the only biotin uptake system that operates in the gut and that its deletion leads to biotin deficiency. Unexpectedly, we also observed that all SMVT-cKO mice develop chronic active inflammation, especially in the cecum. Our aim here was to examine the role of SMVT in the maintenance of intestinal mucosal integrity [permeability and expression of tight junction (TJ) proteins]. Our results showed that knocking out the mouse intestinal SMVT is associated with a significant increase in gut permeability and with changes in the level of expression of TJ proteins. To determine whether these changes are related to the state of biotin deficiency that develops in SMVT-cKO mice, we induced (by dietary means) biotin deficiency in wild-type mice and examined its effect on the above-mentioned parameters. The results showed that dietary-induced biotin deficiency leads to a similar development of chronic active inflammation in the cecum with an increase in the level of expression of proinflammatory cytokines, as well as an increase in intestinal permeability and changes in the level of expression of TJ proteins. We also examined the effect of chronic biotin deficiency on permeability and expression of TJ proteins in confluent intestinal epithelial Caco-2 monolayers but observed no changes in these parameters. These results show that the intestinal SMVT plays an important role in the maintenance of normal mucosal integrity, most likely via its role in providing biotin to different cells of the gut mucosa.

Density functional theory (DFT) has emerged as one of the most versatile and lucrative approaches in electronic structure calculations of many-electron systems in past four decades. Here we give an account of the development of a variational DFT method for atoms and molecules completely in a Cartesian grid. The non-relativistic Kohn–Sham equation is solved by using an LCAO-MO ansatz. Atom-centered localized basis set, electron density, molecular orbitals, two-body potentials are directly constructed on the grid. We adopt a Fourier convolution method for classical Coulomb potentials by making an Ewald-type decomposition technique in terms of short- and long-range interactions. It produces quite accurate and competitive results for various properties of interest, such as component energy, total energy, ionization energy, potential energy curve, atomization energy, etc. Both local and non-local functionals are employed for pseudopotential as well as full calculations. While most results are offered in a uniform grid, initial exploratory attempts are made in a non-uniform grid, which can significantly reduce the computational overhead. This offers a practical, viable alternative to atom-centered grid-based implementations, currently exploited by the majority of programs available world-wide.

Riboflavin (RF) is essential for the normal metabolic activities of pancreatic β-cells and provides protection against oxidative stress. Very little is known about the mechanism of RF uptake by these cells and how the process is regulated. We addressed these issues using mouse-derived pancreatic β-TC-6 cells and freshly isolated primary mouse and human pancreatic islets. Our results showed3H-RF uptake by β-TC-6 cells is Na+independent, cis inhibited by RF-related compounds, trans stimulated by unlabeled RF, and saturable as a function of concentration (apparent Kmof 0.17 ± 0.02 μM). The latter findings suggest involvement of a carrier-mediated process. Similarly, RF uptake by primary mouse and human pancreatic islets was via carrier-mediated process. RF transporters 1, 2, and 3 (RFVT-1, -3, and -2) were all expressed in mouse and human pancreatic β-cells/islets, with RFVT-1 being the predominant transporter expressed in the mouse and RFVT-3 in the human. Specific knockdown of RFVT-1 with gene-specific small interfering RNA leads to a significant inhibition in RF uptake by β-TC-6 cells. RF uptake by β-TC-6 cells was also found to be adaptively upregulated in RF deficiency via a transcriptional mechanism(s). Also, the process appears to be under the regulation of a Ca2+/calmodulin-mediated regulatory pathway. Results of these studies demonstrate, for the first time, the involvement of a carrier-mediated process for RF uptake by mouse and human pancreatic β-cells/islets. Furthermore, the process appears to be regulated by extracellular and intracellular factors.

Colonization factor CS6 of enterotoxigenic Escherichia coli (ETEC) helps to establish the adherence of CS6-expressing ETEC in the intestinal wall. CS6 is composed of two structural subunits, known as CssA and CssB. During CS6-expressing ETEC adherence in intestinal wall, 15 amino acid residues containing Cterminal region of CssA subunit, help to bind with N-terminal 70kDa domain of fibronectin (Fn). In this study, we have predicted a theoretical structural model for C-terminal domain of CssA by homology modelling using protein data bank (PDB) file, 1NTY-A as template (66.67% sequence identity) in Discovery Studio. The structural model of N-terminal region of Fn was also determined by homology modelling using PDB files 1FBR and 1E88 as templates. The structure of the model was also validated by Ramachandran plot. The energy minimization for Fn was performed in standard dynamic cascade using Steepest Descent algorithm followed by Adopted Basis NR algorithm in Discovery studio. The docking model between C-terminal domain and fibronectin were generated by using ClusPro algorithm. This docking study would be help for better understanding how CS6 interacts with fibronectin of intestinal extracellular matrix in the host during infection, and would be of great help towards subunit vaccine generation.

Intestinal absorption of biotin occurs via a Na+-dependent carrier-mediated process that involves the sodium-dependent multivitamin transporter (SMVT; product of the Slc5a6 gene). The SMVT system is exclusively expressed at the apical membrane domain of the polarized intestinal epithelial cells. Whereas previous studies from our laboratory and others have characterized different physiological and biological aspects of SMVT, little is currently known about its structure-function activity relationship. Using site-directed mutagenesis approach, we examined the role of the positively charged histidine (His) residues of the human SMVT (hSMVT) in transporting the negatively charged biotin. Of the seven conserved (across species) His residues in the hSMVT polypeptide, only His115 and His254 were found to be important for the function of hSMVT as their mutation led to a significant reduction in carrier-mediated biotin uptake. This inhibition was mediated via a significant reduction in the maximal velocity ( Vmax), but not the apparent Michaelis constant ( Km), of the biotin uptake process and was not related to the charge of the His residue. The inhibition was also not due to changes in transcriptional or translational efficiency of the mutated hSMVT compared with wild-type carrier. However, surface biotinylation assay showed a significant reduction in the level of expression of the mutated hSMVT at the cell surface, a finding that was further confirmed by confocal imaging. Our results show important role for His115 and His254 residues in hSMVT function, which is most probably mediated via an effect on level of hSMVT expression at the cell membrane.

CS6 is a widely expressed colonization factor of enterotoxigenic Escherichia coli (ETEC). To date, CS6 has not been well characterized in its native state. Here, we purified CS6 for the first time from an ETEC clinical isolate. Purified CS6 was composed of two structural subunits, CssA and CssB, which were present in equal amounts and tightly linked through noncovalent, detergent-stable association. The CssA subunit was poorly immunogenic, whereas CssB was highly immunogenic. Although the predicted molecular mass of CssA is 15 kDa, the purified CssA has an effective molecular mass of 18.5 kDa due to fatty acid modification. When purified CS6 was screened for its ability to bind with different extracellular matrix proteins, fibronectin (Fn) was found to interact with CS6 as well as CssA in a dose-dependent and saturable manner. This interaction was inhibited both by a synthetic peptide corresponding to the C-terminal hydrophilic, surface-exposed region of CssA (positions 112 to 126) and by the antibody derived against this region. Enzyme-linked immunosorbent assay results showed that CssA interacted with the 70-kDa N-terminal domain of Fn. The modifications on CssA probably do not play a role in Fn binding. Preincubation of INT 407 cells with CssA, but not CssB, inhibited ETEC binding to these cells. The results suggested that CS6-expressing ETEC binds to Fn of INT 407 cells through the C-terminal region of CssA. Purified CS6 was found to colocalize with Fn along the junctions of INT 407 cells. Based on the results obtained, we propose that CS6-expressing ETEC binds to the intestinal cells through Fn for colonization.

The chitin-binding protein GbpA of Vibrio cholerae has been recently described as a common adherence factor for chitin and intestinal surface. Using an isogenic in-frame gbpA deletion mutant, we first show that V. cholerae O1 El Tor interacts with mouse intestinal mucus quickly, using GbpA in a specific manner. The gbpA mutant strain showed a significant decrease in intestinal adherence, leading to less colonization and fluid accumulation in a mouse in vivo model. Purified recombinant GbpA (rGbpA) specifically bound to N -acetyl- d -glucosamine residues of intestinal mucin in a dose-dependent, saturable manner with a dissociation constant of 11.2 μM. Histopathology results from infected mouse intestine indicated that GbpA binding resulted in a time-dependent increase in mucus secretion. We found that rGbpA increased the production of intestinal secretory mucins (MUC2, MUC3, and MUC5AC) in HT-29 cells through upregulation of corresponding genes. The upregulation of MUC2 and MUC5AC genes was dependent on NF-κB nuclear translocation. Interestingly, mucin could also increase GbpA expression in V. cholerae in a dose-dependent manner. Thus, we propose that there is a coordinated interaction between GbpA and mucin to upregulate each other in a cooperative manner, leading to increased levels of expression of both of these interactive factors and ultimately allowing successful intestinal colonization and pathogenesis by V. cholerae .

Aims: The aim of this study was to investigate the distribution and inducibility of chitinase genes in vibrios and the effect of environmental factors on the expression level and activity of chitinase genes in Vibrio cholerae strains. Methods and Results: Chitin agar plate assays showed that V. cholerae strains were more chitinolytic than non-cholerae vibrios. All of the identified or putative chitinase genes were expressed in V. cholerae (four strains) but not in non-cholerae vibrios (seven species/strains) under standard laboratory growth conditions. In non-cholerae vibrios, these genes were induced by chitin, its monomer N-acetyl-d-glucosamine and on exposure to rabbit intestine, while in V. cholerae strains, these genes showed significant variation in expression levels. To study the effects of environmental factors on the expression and activity of chitinase genes in V. cholerae, bacteria were cultured in different pH, temperature, sodium chloride and nutrients. RT-PCR analysis showed that lower temperatures and higher pH, salinity and nutrition favoured expression of these genes, while their activity increased under higher nutrition content and salinity. Conclusions: Chitinase genes are distributed in all the relatively small number of strains studied here, and biotic and abiotic factors have significant role in the induction, expression level and activity of this gene family in vibrios. Significance and Impact of the Study: Chitinases have important applications especially in recycling of chitin. Vibrios can be used as chitinolytic agents, using suitable culture conditions that maximize the expression and activity of these genes.

© 2015 Subramanian et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. The intestinal absorption process of vitamin B2 (riboflavin, RF) is carrier-mediated, and all three known human RF transporters, i.e., hRFVT-1, -2, and -3 (products of the SLC52A1, 2 & 3 genes, respectively) are expressed in the gut. We have previously shown that the intestinal RF uptake process is adaptively regulated by substrate level, but little is known about the molecular mechanism(s) involved. Using human intestinal epithelial NCM460 cells maintained under RF deficient and over-supplemented (OS) conditions, we now show that the induction in RF uptake in RF deficiency is associated with an increase in expression of the hRFVT-2 & -3 (but not hRFVT-1) at the protein and mRNA levels. Focusing on hRFVT-3, the predominant transporter in the intestine, we also observed an increase in the level of expression of its hnRNA and activity of its promoter in the RF deficiency state. An increase in the level of expression of the nuclear factor Sp1 (which is important for activity of the SLC52A3 promoter) was observed in RF deficiency, while mutating the Sp1/GC site in the SLC52A3 promoter drastically decreased the level of induction in SLC52A3 promoter activity in RF deficiency. We also observed specific epigenetic changes in the SLC52A3 promoter in RF deficiency. Finally, an increase in hRFVT-3 protein expression at the cell surface was observed in RF deficiency. Results of these investigations show, for the first time, that transcriptional and post-transcriptional mechanisms are involved in the adaptive regulation of intestinal RF uptake by the prevailing substrate level.