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Biomedical subjects

Neerja Karnani

Publications and source records attributed to Neerja Karnani.

10 recordsLinked to original sources
PubMed2026

Secure bioinformatics: privacy-preserving federated analytics using homomorphic encryption.

MOTIVATION: Large-scale bioinformatics analyses increasingly require collaboration across multiple cohorts and institutions, yet existing workflows often rely on data co-localization, which is slow, difficult to scale, and raises privacy concerns. We present a privacy-preserving federated analytics framework that enables secure statistical analysis across distributed datasets without transferring raw data, by performing all computations on encrypted data via cryptographic methods. RESULTS: We evaluate the framework by validating polygenic risk scores and conducting meta-analyses on two real-world cohorts. The proposed solution achieves over 99.9% accuracy relative to plaintext analyses, while maintaining scalable runtime performance with increasing data size and number of participating sites. These results demonstrate the feasibility of secure federated analytics for practical bioinformatics applications involving sensitive data.

Computational Biology↗
PubMed2025

Short-term diet intervention comprising of olive oil, vitamin D, and omega-3 fatty acids alters the small non-coding RNA (sncRNA) landscape of human sperm.

Offspring health outcomes are often linked with epigenetic alterations triggered by maternal nutrition and intrauterine environment. Strong experimental data also link paternal preconception nutrition with pathophysiology in the offspring, but the mechanism(s) routing effects of paternal exposures remain elusive. Animal experimental models have highlighted small non-coding RNAs (sncRNAs) as potential regulators of paternal effects. Here, we characterised the baseline sncRNA landscape of human sperm and the effect of a 6-week dietary intervention on their expression profile. This study involves sncRNAseq profiling, that was performed on a subset (n = 17) of the participants enrolled in the PREPARE trial: 9 from the control group and 8 from the intervention group. 5'tRFs, miRNAs and piRNAs were the most abundant sncRNA subtypes identified; their expression was associated with age, BMI, and sperm quality. Nutritional intervention with olive oil, vitamin D and omega-3 fatty acids altered expression of 3 tRFs, 15 miRNAs and 112 piRNAs, targeting genes involved in fatty acid metabolism and transposable elements in the sperm genome. PREPARE Trial registration number: ISRCTN50956936, Trial registration date: 10/02/2014.

Humans↗
PubMed2005

Temporal profile of replication of human chromosomes.

Chromosomes in human cancer cells are expected to initiate replication from predictably localized origins, firing reproducibly at discrete times in S phase. Replication products obtained from HeLa cells at different stages of S phase were hybridized to cDNA and genome tiling oligonucleotide microarrays to determine the temporal profile of replication of human chromosomes on a genome-wide scale. About 1,000 genes and chromosomal segments were identified as sites containing efficient origins that fire reproducibly. Early replication was correlated with high gene density. An acute transition of gene density from early to late replicating areas suggests that discrete chromatin states dictate early versus late replication. Surprisingly, at least 60% of the interrogated chromosomal segments replicate equally in all quarters of S phase, suggesting that large stretches of chromosomes are replicated by inefficient, variably located and asynchronous origins and forks, producing a pan-S phase pattern of replication. Thus, at least for aneuploid cancer cells, a typical discrete time of replication in S phase is not seen for large segments of the chromosomes.

Chromosomes, Human↗
PubMed2004

Genome-wide expression profile of steroid response in Saccharomyces cerevisiae.

The response of the yeast Saccharomyces cerevisiae to human steroid hormone progesterone was studied by genomic expression profiling. The transcription profile data revealed that steroid response was a global phenomenon wherein a host of genes were affected. For example, 163 genes were upregulated and 40 genes were downregulated, by at least more than twofold. The major categories of upregulated genes included protein destination (15%), metabolism (14%), transport facilitation (12%), cell growth, cell division, and DNA synthesis (8%), and transcription (7%), while metabolism (22%), transcription (11%), intracellular transport (10%), cell growth, cell division, and DNA synthesis (10%), energy (8%), cell rescue, defense, and cell death (6%), and protein synthesis (6%) encoding genes were downregulated. Notwithstanding the fact that yeast cells do not possess commonly occurring steroid response cascade similar to higher eukaryotes, our results demonstrate that a short-term exposure to progesterone results in differential regulation of predominantly stress responsive genes.

Gene Expression Profiling↗
PubMed2004

SRE1 and SRE2 are two specific steroid-responsive modules of Candida drug resistance gene 1 (CDR1) promoter.

CDR1 gene encoding an ATP-driven drug extrusion pump has been implicated in the development of azole-resistance in Candida albicans. Although the upregulation of CDR1 expression by various environmental factors has been documented, the molecular mechanism underlying such process is poorly understood. We have demonstrated earlier that the CDR1 promoter encompasses a large number of cis-regulatory elements, presumably mediating its response to various drugs. In this study we have identified a novel steroid responsive region (SRR) conferring beta-oestradiol and progesterone inducibility on the CDR1 promoter. The SRR is located -696 to -521 bp upstream of the transcription start site; it is modular in nature and can confer steroid responsiveness to a heterologous promoter (ADH1) linked to a GFP reporter gene. In vitro DNase I protection analyses of SRR revealed two progesterone responsive sequences (-628 to -594 and -683 to -648) and one beta-oestradiol responsive sequence (-628 to -577), which was further corroborated by the gel mobility shift assay. Deletion analyses within the SRR further delimited these steroid responsive sequences into two distinct elements, viz. SRE1 and SRE2. While SRE1 (-677 to -648) responds only to progesterone, SRE2 (-628 to -598) responded to both progesterone and beta-oestradiol. Both SRE1 and SRE2 were specific for steroids, as they did not respond to other drugs, such as cycloheximide, miconazole and terbinafine. In silico comparison of the SRE1/2 with the promoter sequences of other MDR (CDR2 and PDR5) and non-MDR (HSP90) steroid-responsive genes revealed a similarity with respect to conservation of three 5 bp stretches (AAGAA, CCGAA and ATTGG). Taken together, we have identified a novel steroid responsive cis-regulatory sequence in the CDR1 promoter, which presumably can be instrumental in understanding the steroid response cascade in Candida albicans.

Antifungal Agents↗
PubMed2004

ABC multidrug transporter Cdr1p of Candida albicans has divergent nucleotide-binding domains which display functional asymmetry.

In order to ascertain the molecular basis of ATP-mediated drug extrusion by Cdr1p, a multidrug transporter of Candida albicans, we recently have reported that the Walker A motif of the N-terminal nucleotide biding domain (NBD) of this protein contains an uncommon cysteine residue (C193; GXXGXGCS/T) which is indispensable for ATP hydrolysis. This residue is exceptionally conserved in N-terminal NBDs of fungal ABC transporters and hence makes these transporters an evolutionarily divergent group. However, the presence of a conventional lysine residue at a similar position in the Walker A motif of the C-terminal NBD warrants the individual contribution of both the NBDs in the ATP-driven efflux function of such transporters. In this study we have investigated the contribution of this divergent Walker A motif in the context of the full Cdr1p protein under in vivo conditions by swapping these two crucial amino acids (C193K in Walker A motif of N-terminal NBD and K901C in Walker A motif of C-terminal NBD) between the two NBDs. Both the native and the mutant variants of Cdr1p were integrated at the PDR5 locus as GFP-tagged fusion proteins and were hyper-expressed. Our study shows that both C193K- and K901C-expressing cells elicit a severe impairment of Cdr1p's ATPase function. However, both these mutations have distinct phenotypes with respect to other functional parameters such as substrate efflux and drug resistance profiles. In contrast to C193K, K901C mutant cells were substantially hypersensitive to the tested drugs (fluconazole, ansiomycin, miconazole and cycloheximide) and were unable to expel rhodamine 6G. Our results for the first time show that both NBDs influence the Cdr1p function asymmetrically, and that the positioning of the cysteine and lysine residues within the respective Walker A motifs is functionally not interchangeable.

Adenosine Triphosphatases↗
PubMed2004

Identification of a negative regulatory element which regulates basal transcription of a multidrug resistance gene CDR1 of Candida albicans.

We have earlier shown that transcriptional activation of the Candida drug resistance gene, CDR1, is linked to various stresses wherein a proximal promoter (-345 bp from the transcription start point (TSP)) was found to be predominantly more responsive. In this study we have examined basal expression of the CDR1 proximal promoter by employing a Renilla luciferase reporter system. We observed that upon sequential deletion of the proximal promoter, there was modulation in basal reporter activity. The reporter activity was highest (2.3-fold) in NGY261 (-261 bp from TSP), and was reduced upon subsequent deletions. DNase I footprinting revealed four protected regions (W1, W2, W3 and W4) in the proximal promoter which could represent possible trans-acting factor binding sites and thus might be involved in CDR1 expression. Site-directed mutational analysis of three of these protected regions did not significantly affect the basal reporter activity, however, the mutation of W1 led to a considerable enhancement in reporter activity (approximately 4-fold) and was designated a negative regulatory element (NRE). Mutation as well as deletion of the W1 sequence in the native promoter (-1147 bp from TSP) and sequential deletion of the 5'-flanking region-harboring W1 (NRE) also resulted in enhanced promoter reporter activity. When the reporter activity of native (NPY1147) and NRE-mutated (NGYM1147) promoter integrants was monitored throughout the growth phase of Candida albicans, there was modulation in reporter activity in both integrants, but interestingly the level of basal reporter activity of the NRE-mutated promoter was always approximately 3-fold higher than that of the native promoter. UV cross-linking and affinity purification confirmed that a purified approximately 55-kDa nuclear protein specifically interacts with the NRE. Taken together, we have identified a NRE and purified its interactive protein, which may be involved in controlling basal expression of CDR1.

Base Sequence↗
PubMed2003

Covalent modification of cysteine 193 impairs ATPase function of nucleotide-binding domain of a Candida drug efflux pump.

N-ethylmaleimide (NEM) impairs the ATPase function of N-terminal NBD of Candida drug resistance gene product Cdr1p. To identify the reactive cysteine(s) for such a contribution, we adopted a three-arm approach that included covalent modification, cysteine mutagenesis, and structure homology modeling. The covalent modification results clearly indicate the ability of NEM and iodoacetic acid (IAA) to potently inhibit the ATPase activity of N-terminal NBD. Since this domain contains five cysteine residues in its sequence, we mutated each and found four of these (C325A, C363A, C402A, and C462A) to stay sensitive to NEM/IAA modification and influence ATPase activity, while C193A mutation completely abrogated the catalytic function. The structural homology modeling data further validate these biochemical findings by ruling out any plausible interactions within the cysteine residues, and deriving the importance of Cys-193 in lying at a bond length clearly feasible to interact with ATP and divalent cation to critically influence ATP hydrolysis.

Adenosine Triphosphatases↗
PubMed2003

Purification and characterization of the N-terminal nucleotide binding domain of an ABC drug transporter of Candida albicans: uncommon cysteine 193 of Walker A is critical for ATP hydrolysis.

The Candida drug resistance protein Cdr1p (approximately 170 kDa) is a member of ATP binding cassette (ABC) superfamily of drug transporters, characterized by the presence of 2 nucleotide binding domains (NBD) and 12 transmembrane segments (TMS). NBDs of these transporters are the hub of ATP hydrolysis activity, and their sequence contains a conserved Walker A motif (GxxGxGKS/T). Mutations of the lysine residue within this motif abrogate the ability of NBDs to hydrolyze ATP. Interestingly, the sequence alignments of Cdr1p NBDs with other bacterial and eukaryotic transporters reveal that its N-terminal NBD contains an unusual Walker A sequence (GRPGAGCST), as the invariant lysine is replaced by a cysteine. In an attempt to understand the significance of this uncommon positioning of cysteine within the Walker A motif, we for the first time have purified and characterized the N-terminal NBD (encompassing first N-terminal 512 amino acids) of Cdr1p as well as its C193A mutant protein. The purified NBD-512 protein could exist as an independent functional general ribonucleoside triphosphatase with strong divalent cation dependence. It exhibited ATPase activity with an apparent K(m) in the 0.8-1.0 mM range and V(max) in the range of 147-160 nmol min(-)(1) (mg of protein)(-)(1). NBD-512-associated ATPase activity was also sensitive to inhibitors such as vanadate, azide, and NEM. The Mut-NBD-512 protein (C193A) showed a severe impairment in its ability to hydrolyze ATP (95%); however, no significant effect on ATP (TNP-ATP) binding was observed. Our results show that C193 is critical for N-terminal NBD-mediated ATP hydrolysis and represents a unique feature distinguishing the ATP-dependent functionality of the ABC transporters of fungi from those found in bacteria and other eukaryotes.

ATP-Binding Cassette Transporters↗