Evidence for clustered mannose as a new ligand for hyaluronan-binding protein (HABP1) from human fibroblasts

We have earlier reported that overexpression of the gene encoding human hyaluronan-binding protein (HABP1) is functionally active, as it binds specifically with hyaluronan (HA). In this communication, we confirm the collapse of the filamentous and branched structure of HA by interaction with increasing concentrations of recombinant-HABP1 (rHABP1). HA is the reported ligand of rHABP1. Here, we show the affinity of rHABP1 towards D-mannosylated albumin (DMA) by overlay assay and purification using a DMA affinity column. Our data suggests that DMA is another ligand for HABP1. Furthermore, we have observed that DMA inhibits the binding of HA in a concentration-dependent manner, suggesting its multiligand affinity amongst carbohydrates. rHABP1 shows differential affinity towards HA and DMA which depends on pH and ionic strength. These data suggest that affinity of rHABP1 towards different ligands is regulated by the microenvironment

Functional characterization of Helicobacter pylori DnaB helicase

Helicobacter pylori causes gastric ulcer diseases and gastric adenocarcinoma in humans. Not much is known regarding DNA replication in H. pylori that is important for cell survival. Here we report the cloning, expression and characterization of H. pylori DnaB (HpDnaB) helicase both in vitro and in vivo. Among the DnaB homologs, only Escherichia coli DnaB has been studied extensively. HpDnaB showed strong 5 to 3′ helicase and ATPase activity. Interestingly, H. pylori does not have an obvious DnaC homolog which is essential for DnaB loading on the E. coli chromosomal DNA replication origin (oriC). However, HpDnaB can functionally complement the E. coli DnaB temperature‐sensitive mutant at the non‐permissive temperature, confirming that HpDnaB is a true replicative helicase. Escherichia coli DnaC co‐eluted in the same fraction with HpDnaB following gel filtration analysis suggesting that these proteins might physically interact with each other. It is possible that a functional DnaC homolog is present in H. pylori. The complete characterization of H. pylori DnaB helicase will also help the comparative analysis of DnaB helicases among bacteria

Characterisation and mode of in vitro replication of pea chloroplast OriA sequences

A partially purified replicative system of pea chloroplast that replicates recombinant DNAs containing pea chloroplast origin sequences has been characterised. Polymerisation by this system is very fast and insensitive to chain terminators like dideoxynucleotides, arabinosylcytosine 5'-triphosphate, etc. Both strands of template DNA are synthesized and single-stranded DNA templates undergo more than one round of replication. When sequences of either of the two chloroplast origins of replication (OriA or OriB) are used as templates, the replicative intermediates are found to have sigma structures. Electron microscopic analysis of the sigma structures restricted with various enzymes reveals that the initiation site of in vitro replication maps near the displacement-loop regions where replication initiates also in vivo. Although the observed replication initiation in the OriA recombinant template is chloroplast-DNA-specific, the mode of replication is different from that observed in vivo with intact ctDNA. However, when the template DNA contains both the OriA and OriB sequences, the in vitro replication proceeds in the theta mode, the mode of replication usually observed in vivo

Detailed study of the ELAIS N1 field with the uGMRT - I. Characterizing the 325 MHz foreground for redshifted 21 cm observations

In this first paper of the series, we present initial results of newly upgraded Giant Meterwave Radio Telescope (uGMRT) observation of European Large-Area ISO Survey-North 1 (ELAIS-N1) at 325 MHz with 32 MHz bandwidth. Precise measurement of fluctuations in Galactic and extragalactic foreground emission as a function of frequency as well as angular scale is necessary for detecting redshifted 21-cm signal of neutral hydrogen from Cosmic Dawn, Epoch of Reionization (EoR) and post-reionization epoch. Here, for the first time we have statistically quantified the Galactic and extragalactic foreground sources in the ELAIS-N1 field in the form of angular power spectrum using the newly developed Tapered Gridded Estimator (TGE). We have calibrated the data with and without direction-dependent calibration techniques. We have demonstrated the effectiveness of TGE against the direction dependent effects by using higher tapering of field of view (FoV). We have found that diffuse Galactic synchrotron emission (DGSE) dominates the sky, after point source subtraction, across the angular multipole range $1115 \leqslant \mathcal{\ell} \leqslant 5083$ and $1565 \leqslant \mathcal{\ell} \leqslant 4754$ for direction-dependent and -independent calibrated visibilities respectively. The statistical fluctuations in DGSE has been quantified as a power law of the form $\mathcal{C}_{\mathcal{\ell}}= A \mathcal{\ell}^{-\beta}$. The best fitted values of (A, $\beta$) are ($62 \pm 6$ $mK^{2}$, $2.55 \pm 0.3$) and ($48 \pm 4$ $mK^{2}$, $2.28 \pm 0.4$ ) for the two different calibration approaches. For both the cases, the power law index is consistent with the previous measurements of DGSE in other parts of sky.Comment: 13 pages, 5figures, 4 tables; accepted for publication in MNRA

The oligomeric Rep protein of Mungbean yellow mosaic India virus (MYMIV) is a likely replicative helicase

Geminiviruses replicate by rolling circle mode of replication (RCR) and the viral Rep protein initiates RCR by the site-specific nicking at a conserved nonamer (TAATATT↓ AC) sequence. The mechanism of subsequent steps of the replication process, e.g. helicase activity to drive fork-elongation, etc. has largely remained obscure. Here we show that Rep of a geminivirus, namely, Mungbean yellow mosaic India virus (MYMIV), acts as a replicative helicase. The Rep-helicase, requiring ≥6 nt space for its efficient activity, translocates in the 3'→5' direction, and the presence of forked junction in the substrate does not influence the activity to any great extent. Rep forms a large oligomeric complex and the helicase activity is dependent on the oligomeric conformation (∼24mer). The role of Rep as a replicative helicase has been demonstrated through ex vivo studies in Saccharomyces cerevisiae and in planta analyses in Nicotiana tabacum. We also establish that such helicase activity is not confined to the MYMIV system alone, but is also true with at least two other begomoviruses, viz., Mungbean yellow mosaic virus (MYMV) and Indian cassava mosaic virus (ICMV)

Towards 21-cm Intensity Mapping at z=2.28z=2.28 with uGMRT using the Tapered Gridded Estimator I: Foreground Avoidance

The post-reionization $(z \le 6)$ neutral hydrogen (HI) 21-cm intensity mapping signal holds the potential to probe the large scale structures, study the expansion history and constrain various cosmological parameters. Here we apply the Tapered Gridded Estimator (TGE) to estimate $P(k_{\perp},k_{\parallel})$ the power spectrum of the $z = 2.28$ $(432.8\, {\rm MHz})$ redshifted 21-cm signal using a $24.4\,{\rm MHz}$ sub-band drawn from uGMRT Band 3 observations of European Large-Area ISO Survey-North 1 (ELAIS-N1). The TGE allows us to taper the sky response which suppresses the foreground contribution from sources in the periphery of the telescope's field of view. We apply the TGE on the measured visibility data to estimate the multi-frequency angular power spectrum (MAPS) $C_{\ell}(\Delta\nu)$ from which we determine $P(k_{\perp},k_{\parallel})$ using maximum-likelihood which naturally overcomes the issue of missing frequency channels (55 \% here). The entire methodology is validated using simulations. For the data, using the foreground avoidance technique, we obtain a $2\,\sigma$ upper limit of $\Delta^2(k) \le (133.97)^2 \, {\rm mK}^{2}$ for the 21-cm brightness temperature fluctuation at $k = 0.347 \, \textrm{Mpc}^{-1}$. This corresponds to $[\Omega_{\rm HI}b_{\rm HI}] \le 0.23$, where $\Omega_{\rm HI}$ and $b_{\rm HI}$ respectively denote the cosmic \HI mass density and the \HI bias parameter. A previous work has analyzed $8 \, {\rm MHz}$ of the same data at $z=2.19$, and reported $\Delta^{2}(k) \le (61.49)^{2} \, {\rm mK}^{2}$ and $[\Omega_{\rm HI} b_{\rm HI}] \le 0.11$ at $k=1 \, {\rm Mpc}^{-1}$. The upper limits presented here are still orders of magnitude larger than the expected signal corresponding to $\Omega_{\rm HI} \sim 10^{-3}$ and $b_{\rm HI} \sim 2$.Comment: 13 pages, 11 figures, accepted for publication in MNRA

Towards 2121-cm intensity mapping at z=2.28z=2.28 with uGMRT using the tapered gridded estimator III: Foreground removal

Neutral hydrogen (\ion{H}{i}) $21$-cm intensity mapping (IM) is a promising probe of the large-scale structures in the Universe. However, a few orders of magnitude brighter foregrounds obscure the IM signal. Here we use the Tapered Gridded Estimator (TGE) to estimate the multi-frequency angular power spectrum (MAPS) $C_{\ell}(\Delta\nu)$ from a $24.4\,\rm{MHz}$ bandwidth uGMRT Band $3$ data at $432.8\,\rm{MHz}$. In $C_{\ell}(\Delta\nu)$ foregrounds remain correlated across the entire $\Delta\nu$ range, whereas the $21$-cm signal is localized within $\Delta\nu\le[\Delta \nu]$ (typically $0.5-1\,\rm{MHz}$). Assuming the range $\Delta\nu>[\Delta \nu]$ to have minimal $21$-cm signal, we use $C_{\ell}(\Delta\nu)$ in this range to model the foregrounds. This foreground model is extrapolated to $\Delta\nu\leq[\Delta \nu]$, and subtracted from the measured $C_{\ell}(\Delta\nu)$. The residual $[C_{\ell}(\Delta\nu)]_{\rm res}$ in the range $\Delta\nu\le[\Delta\nu]$ is used to constrain the $21$-cm signal, compensating for the signal loss from foreground subtraction. $[C_{\ell}(\Delta\nu)]_{\rm{res}}$ is found to be noise-dominated without any trace of foregrounds. Using $[C_{\ell}(\Delta\nu)]_{\rm res}$ we constrain the $21$-cm brightness temperature fluctuations $\Delta^2(k)$, and obtain the $2\sigma$ upper limit $\Delta_{\rm UL}^2(k)\leq(18.07)^2\,\rm{mK^2}$ at $k=0.247\,\rm{Mpc}^{-1}$. We further obtain the $2\sigma$ upper limit [\Omega_{\ion{H}{i}}b_{\ion{H}{i}}]_{\rm UL}\leq0.022 where \Omega_{\ion{H}{i}} and b_{\ion{H}{i}} are the comoving \ion{H}{i} density and bias parameters respectively. Although the upper limit is nearly $10$ times larger than the expected $21$-cm signal, it is $3$ times tighter over previous works using foreground avoidance on the same data.Comment: Accepted for publication in MNRAS. 16 pages (including Appendix), 8 figures (plus 8 in Appendix), 5 Table

Single-stranded DNA binding protein from human malarial parasite Plasmodium falciparum is encoded in the nucleus and targeted to the apicoplast

Apicoplast, an essential organelle of human malaria parasite Plasmodium falciparum contains a ∼35 kb circular genome and is a possible target for therapy. Proteins required for the replication and maintenance of the apicoplast DNA are not clearly known. Here we report the presence of single–stranded DNA binding protein (SSB) in P falciparum. PfSSB is targeted to the apicoplast and it binds to apicoplast DNA. A strong ssDNA binding activity specific to SSB was also detected in P. falciparum lysate. Both the recombinant and endogenous proteins form tetramers and the homology modelling shows the presence of an oligosaccharide/oligonucleotide-binding fold responsible for ssDNA binding. Additionally, we used SSB as a tool to track the mechanism of delayed death phenomena shown by apicoplast targeted drugs ciprofloxacin and tetracycline. We find that the transport of PfSSB is severely affected during the second life cycle following drug treatment. Moreover, the translation of PfSSB protein and not the transcription of PfSSB seem to be down-regulated specifically during second life cycle although there is no considerable change in protein expression profile between drug-treated and untreated parasites. These results suggest dual control of translocation and translation of apicoplast targeted proteins behind the delayed death phenomena