Prevalence and Determinants of Low Birth Weight among the Newborns in Dadra and Nagar Haveli: A Community-based Study.

Background: The world is not on track to meet the World Health Assembly (WHA) global target on Low Birth Weight (LBW). To estimate the prevalence and to identify the associated determinants of LBW among the newborns. Material and Methods: We conducted a cross-sectional study among the 364 mothers registered under the all government health facilities of Dadra & Nagar Haveli (DNH) during November 2021 to January 2022. Results: The prevalence of LBW was found to be 39%. On uni-variable logistic regression, live in relationship, caste, weight of mother, Body Mass Index (BMI), weight gain <5 kg in 2nd and 3rd trimester, high-risk pregnancy, complication present in previous pregnancy and preterm delivery, while on multi-variable logistic regression, weight gain <5 kg in 2nd and 3rd trimester (AOR 2, 95% CI 1.007-4.2) and having high-risk pregnancy (AOR 2, 95% CI 1.1-3.0) were found to be the significant predictors of LBW among the newborns. Conclusions: We conclude from the study that the prevalence of low birth weight among the newborn was high. There is a need to address maternal and child health issues like low birth weight, malnutrition and high-risk pregnancy under the RMNCAH+N program through various effective interventions. Future research should evaluate the feasibility of collaborative activities between RMNCAH+N program and the UNICEF in India.

High-throughput screening of compounds targeting RNA editing in Trypanosoma brucei: Novel molecular scaffolds with broad trypanocidal effects.

Mitochondrial uridine insertion/deletion RNA editing, catalyzed by a multiprotein complex (editosome), is essential for gene expression in trypanosomes and Leishmania parasites. As this process is absent in the human host, a drug targeting this mechanism promises high selectivity and reduced toxicity. Here, we successfully miniaturized our FRET-based full-round RNA editing assay, which replicates the complete RNA editing process, adapting it into a 1536-well format. Leveraging this assay, we screened over 100,000 compounds against purified editosomes derived from Trypanosoma brucei, identifying seven confirmed primary hits. We sourced and evaluated various analogs to enhance the inhibitory and parasiticidal effects of these primary hits. In combination with secondary assays, our compounds marked inhibition of essential catalytic activities, including the RNA editing ligase and interactions of editosome proteins. Although the primary hits did not exhibit any growth inhibitory effect on parasites, we describe eight analog compounds capable of effectively killing T. brucei and/or Leishmania donovani parasites within a low micromolar concentration. Whether parasite killing is - at least in part - due to inhibition of RNA editing in vivo remains to be assessed. Our findings introduce novel molecular scaffolds with the potential for broad antitrypanosomal effects.

The discovery and characterization of two novel structural motifs on the carboxy-terminal domain of kinetoplastid RNA editing ligases.

Parasitic protozoans of the Trypanosoma and Leishmania species have a uniquely organized mitochondrial genome, the kinetoplast. Most kinetoplast-transcribed mRNAs are cryptic and encode multiple subunits for the electron transport chain following maturation through a uridine insertion/deletion process called RNA editing. This process is achieved through an enzyme cascade by an RNA editing catalytic complex (RECC), where the final ligation step is catalyzed by the kinetoplastid RNA editing ligases, KREL1 and KREL2. While the amino-terminal domain (NTD) of these proteins is highly conserved with other DNA ligases and mRNA capping enzymes, with five recognizable motifs, the functional role of their diverged carboxy-terminal domain (CTD) has remained elusive. In this manuscript, we assayed recombinant KREL1 in vitro to unveil critical residues from its CTD to be involved in protein-protein interaction and dsRNA ligation activity. Our data show that the α-helix (H)3 of KREL1 CTD interacts with the αH1 of its editosome protein partner KREPA2. Intriguingly, the OB-fold domain and the zinc fingers on KREPA2 do not appear to influence the RNA ligation activity of KREL1. Moreover, a specific KWKE motif on the αH4 of KREL1 CTD is found to be implicated in ligase auto-adenylylation analogous to motif VI in DNA ligases. In summary, we present in the KREL1 CTD a motif VI for auto-adenylylation and a KREPA2 binding motif for RECC integration.

Hammerhead ribozyme-based U-insertion and deletion RNA editing assays for multiplexing in HTS applications.

Untranslatable mitochondrial transcripts in kinetoplastids are decrypted post-transcriptionally through an RNA editing process that entails uridine insertion/deletion. This unique stepwise process is mediated by the editosome, a multiprotein complex that is a validated drug target of considerable interest in addressing the unmet medical needs for kinetoplastid diseases. With that objective, several in vitro RNA editing assays have been developed, albeit with limited success in discovering potent inhibitors. This manuscript describes the development of three hammerhead ribozyme (HHR) FRET reporter-based RNA editing assays for precleaved deletion, insertion, and ligation assays that bypass the rate-limiting endonucleolytic cleavage step, providing information on U-deletion, U-insertion, and ligation activities. These assays exhibit higher editing efficiencies in shorter incubation times while requiring significantly less purified editosome and 10,000-fold less ATP than the previously published full round of in vitro RNA editing assay. Moreover, modifications in the reporter ribozyme sequence enable the feasibility of multiplexing a ribozyme-based insertion/deletion editing (RIDE) assay that simultaneously surveils U-insertion and deletion editing suitable for HTS. These assays can be used to find novel chemical compounds with chemotherapeutic applications or as probes for studying the editosome machinery.

Sulfonated inhibitors of the RNA editing ligases validate the essential role of the MRP1/2 proteins in kinetoplastid RNA editing.

The RNA editing core complex (RECC) catalyzes mitochondrial U-insertion/deletion mRNA editing in trypanosomatid flagellates. Some naphthalene-based sulfonated compounds, such as C35 and MrB, competitively inhibit the auto-adenylylation activity of an essential RECC enzyme, kinetoplastid RNA editing ligase 1 (KREL1), required for the final step in editing. Previous studies revealed the ability of these compounds to interfere with the interaction between the editosome and its RNA substrates, consequently affecting all catalytic activities that comprise RNA editing. This observation implicates a critical function for the affected RNA binding proteins in RNA editing. In this study, using the inhibitory compounds, we analyzed the composition and editing activities of functional editosomes and identified the mitochondrial RNA binding proteins 1 and 2 (MRP1/2) as their preferred targets. While the MRP1/2 heterotetramer complex is known to bind guide RNA and promote annealing to its cognate pre-edited mRNA, its role in RNA editing remained enigmatic. We show that the compounds affect the association between the RECC and MRP1/2 heterotetramer. Furthermore, RECC purified post-treatment with these compounds exhibit compromised in vitro RNA editing activity that, remarkably, recovers upon the addition of recombinant MRP1/2 proteins. This work provides experimental evidence that the MRP1/2 heterotetramer is required for in vitro RNA editing activity and substantiates the hypothesized role of these proteins in presenting the RNA duplex to the catalytic complex in the initial steps of RNA editing.

Scale Effect of a Fluorescent Waveguide in Organic Micromaterials: A Case Study Based on Coumarin Microfibers.

The classical method for evaluating the waveguide ability only focuses on the optical loss coefficient. However, for the micro- or submicroscale, an organic waveguide is demonstrated by the present study whose scale effect should not be neglected. We found that the optical loss coefficient increased remarkably when decreasing the sectional size of the microfibers. Furthermore, simulations based on Finite-Difference Time-Domain also demonstrated the size-dependent effect of the waveguide. Both the experimental and simulating results showed that the optical loss coefficient converges to a certain value, which means that the scale effect can be neglected as the sectional size is large enough. On the basis of the present study, we suggest that the scale-dependent effect on the sectional size of the waveguide should be investigated by evaluating the waveguide ability by the optical loss coefficient.

Pd-catalyzed cascade reactions involving skipped dienes: from double carbopalladation to remote C-C cleavage.

We report two ligand-controlled cascade reactions relying on the intramolecular carbopalladation of skipped dienes. The use of a bulky monodentate phosphine ligand affords [4,5]-spirocycles via sequential double carbopalladation, however bidentate phosphines promote a remote ß-C-elimination process which does not rely on the use of strained or sterically hindered substrates.

The interaction of a Trypanosoma brucei KH-domain protein with a ribonuclease is implicated in ribosome processing.

Ribosomal RNA maturation is best understood in yeast. While substantial efforts have been made to explore parts of these essential pathways in animals, the similarities and uniquenesses of rRNA maturation factors in non-Opisthokonts remain largely unexplored. Eukaryotic ribosome synthesis requires the coordinated activities of hundreds of Assembly Factors (AFs) that transiently associate with pre-ribosomes, many of which are essential. Pno1 and Nob1 are two of six AFs that are required for the cytoplasmic maturation of the 20S pre-rRNA to 18S rRNA in yeast where it has been almost exclusively analyzed. Specifically, Nob1 ribonucleolytic activity generates the mature 3'-end of 18S rRNA. We identified putative Pno1 and Nob1 homologues in the protist Trypanosoma brucei, named TbPNO1 and TbNOB1, and set out to explore their rRNA maturation role further as they are both essential for normal growth. TbPNO1 is a nuclear protein with limited cytosolic localization relative to its yeast homologue. Like in yeast, it interacts directly with TbNOB1, with indications of associations with a larger AF-containing complex. Interestingly, in the absence of TbPNO1, TbNOB1 exhibits non-specific degradation activity on RNA substrates, and its cleavage activity becomes specific only in the presence of TbPNO1, suggesting that TbPNO1-TbNOB1 interaction is essential for regulation and site-specificity of TbNOB1 activity. These results highlight a conserved role of the TbPNO1-TbNOB1 complex in 18S rRNA maturation across eukaryotes; yet reveal a novel role of their interaction in regulation of TbNOB1 enzymatic activity.

A Protein Complex Map of Trypanosoma brucei.

The functions of the majority of trypanosomatid-specific proteins are unknown, hindering our understanding of the biology and pathogenesis of Trypanosomatida. While protein-protein interactions are highly informative about protein function, a global map of protein interactions and complexes is still lacking for these important human parasites. Here, benefiting from in-depth biochemical fractionation, we systematically interrogated the co-complex interactions of more than 3354 protein groups in procyclic life stage of Trypanosoma brucei, the protozoan parasite responsible for human African trypanosomiasis. Using a rigorous methodology, our analysis led to identification of 128 high-confidence complexes encompassing 716 protein groups, including 635 protein groups that lacked experimental annotation. These complexes correlate well with known pathways as well as for proteins co-expressed across the T. brucei life cycle, and provide potential functions for a large number of previously uncharacterized proteins. We validated the functions of several novel proteins associated with the RNA-editing machinery, identifying a candidate potentially involved in the mitochondrial post-transcriptional regulation of T. brucei. Our data provide an unprecedented view of the protein complex map of T. brucei, and serve as a reliable resource for further characterization of trypanosomatid proteins. The presented results in this study are available at: www.TrypsNetDB.org.

Mutational analysis of Trypanosoma brucei RNA editing ligase reveals regions critical for interaction with KREPA2.

The Trypanosoma brucei parasite causes the vector-borne disease African sleeping sickness. Mitochondrial mRNAs of T. brucei undergo posttranscriptional RNA editing to make mature, functional mRNAs. The final step of this process is catalyzed by the essential ligase, T. brucei RNA Editing Ligase 1 (TbREL1) and the closely related T. brucei RNA Editing Ligase 2 (TbREL2). While other ligases such as T7 DNA ligase have both a catalytic and an oligonucleotide/oligosaccharide-binding (OB)-fold domain, T. brucei RNA editing ligases contain only the catalytic domain. The OB-fold domain, which is required for interaction with the substrate RNA, is provided in trans by KREPA2 (for TbREL1) and KREPA1 (for TbREL2). KREPA2 enhancement of TbREL1 ligase activity is presumed to occur via an OB-fold-mediated increase in substrate specificity and catalysis. We characterized the interaction between TbREL1 and KREPA2 in vitro using full-length, truncated, and point-mutated ligases. As previously shown, our data indicate strong, specific stimulation of TbREL1 catalytic activity by KREPA2. We narrowed the region of contact to the final 59 C-terminal residues of TbREL1. Specifically, the TbREL1 C-terminal KWKE (441-444) sequence appear to coordinate the KREPA2-mediated enhancement of TbREL1 activities. N-terminal residues F206, T264 and Y275 are crucial for the overall activity of TbREL1, particularly for F206, a mutation of this residue also disrupts KREPA2 interaction. Thus, we have identified the critical TbREL1 regions and amino acids that mediate the KREPA2 interaction.

Pilot-scale compound screening against RNA editing identifies trypanocidal agents.

Most mitochondrial messenger RNAs in trypanosomatid pathogens undergo a unique type of posttranscriptional modification involving insertion and/or deletion of uridylates. This process, RNA editing, is catalyzed by a multiprotein complex (~1.6 MDa), the editosome. Knockdown of core editosome proteins compromises mitochondrial function and, ultimately, parasite viability. Hence, because the editosome is restricted to trypanosomatids, it serves as a unique drug target in these pathogens. Currently, there is a lack of editosome inhibitors for antitrypanosomatid drug development or that could serve as unique tools for perturbing and characterizing editosome interactions or RNA editing reaction stages. Here, we screened a library of pharmacologically active compounds (LOPAC1280) using high-throughput screening to identify RNA editing inhibitors. We report that aurintricarboxylic acid, mitoxantrone, PPNDS, and NF449 are potent inhibitors of deletion RNA editing (IC50 range, 1-5 µM). However, none of these compounds could specifically inhibit the catalytic steps of RNA editing. Mitoxantrone blocked editing by inducing RNA-protein aggregates, whereas the other three compounds interfered with editosome-RNA interactions to varying extents. Furthermore, NF449, a suramin analogue, was effective at killing Trypanosoma brucei in vitro. Thus, new tools for editosome characterization and downstream RNA editing inhibitor have been identified.

RNA catalyst as a reporter for screening drugs against RNA editing in trypanosomes.

Substantial progress has been made in determining the mechanism of mitochondrial RNA editing in trypanosomes. Similarly, considerable progress has been made in identifying the components of the editosome complex that catalyze RNA editing. However, it is still not clear how those proteins work together. Chemical compounds obtained from a high-throughput screen against the editosome may block or affect one or more steps in the editing cycle. Therefore, the identification of new chemical compounds will generate valuable molecular probes for dissecting the editosome function and assembly. In previous studies, in vitro editing assays were carried out using radio-labeled RNA. These assays are time consuming, inefficient and unsuitable for high-throughput purposes. Here, a homogenous fluorescence-based "mix and measure" hammerhead ribozyme in vitro reporter assay to monitor RNA editing, is presented. Only as a consequence of RNA editing of the hammerhead ribozyme a fluorescence resonance energy transfer (FRET) oligoribonucleotide substrate undergoes cleavage. This in turn results in separation of the fluorophore from the quencher thereby producing a signal. In contrast, when the editosome function is inhibited, the fluorescence signal will be quenched. This is a highly sensitive and simple assay that should be generally applicable to monitor in vitro RNA editing or high throughput screening of chemicals that can inhibit the editosome function.

Ruthenium-catalyzed cascade C-H functionalization of phenylacetophenones.

Three orthogonal cascade CH functionalization processes are described, based on ruthenium-catalyzed CH alkenylation. 1-Indanones, indeno indenes, and indeno furanones were accessed through cascade pathways by using arylacetophenones as substrates under conditions of catalytic [{Ru(p-cymene)Cl2 }2 ] and stoichiometric Cu(OAc)2 . Each transformation uses CH functionalization methods to form CC bonds sequentially, with the indeno furanone synthesis featuring a CO bond formation as the terminating step. This work demonstrates the power of ruthenium-catalyzed alkenylation as a platform reaction to develop more complex transformations, with multiple CH functionalization steps taking place in a single operation to access novel carbocyclic structures.

S-, N-, and Se-difluoromethylation using sodium chlorodifluoroacetate.

A simple protocol for the difluoromethylation of thiols is reported using chlorodifluoroacetate as the difluoromethylating agent. This cheap reagent undergoes smooth decarboxylation at 95 °C to afford difluorocarbene, which can be trapped with a variety of aromatic and heteroaromatic thiols. The reaction is also effective for the difluoromethylation of heterocyclic nitrogen compounds and phenylselenol.

Global identification of conserved post-transcriptional regulatory programs in trypanosomatids.

While regulatory programs are extensively studied at the level of transcription, elements that are involved in regulation of post-transcriptional processes are largely unknown, and methods for systematic identification of these elements are in early stages. Here, using a novel computational framework, we have integrated sequence information with several functional genomics data sets to characterize conserved regulatory programs of trypanosomatids, a group of eukaryotes that almost entirely rely on post-transcriptional processes for regulation of mRNA abundance. This analysis revealed a complex network of linear and structural RNA elements that potentially govern mRNA abundance across different life stages and environmental conditions. Furthermore, we show that the conserved regulatory network that we have identified is responsive to chemical perturbation of several biological functions in trypanosomatids. We have further characterized one of the most abundant regulatory RNA elements that we discovered, an AU-rich element (ARE) that can be found in 3' untranslated region of many trypanosomatid genes. Using bioinformatics approaches as well as in vitro and in vivo experiments, we have identified three ELAV-like homologs, including the developmentally critical protein TbRBP6, which regulate abundance of a large number of trypanosomatid ARE-containing transcripts. Together, these studies lay out a roadmap for characterization of mechanisms that modulate development and metabolic pathways in trypanosomatids.

Transition metal-catalyzed C-C bond formation via C-S bond cleavage: an overview.

Transition metal-catalyzed C-C bond formations have been well studied over the last four decades. An improved mechanistic understanding of such reactions has helped chemists to develop further improvements, modifications and even new reactions. In the area of transition metal-catalyzed cross-coupling reactions the C-S bond cleaving reactions have attracted a lot of attention in the last decade as they provide a good alternative to the use of organo-halide reagents in traditional cross-coupling reactions. The availability of a wide range of organo-sulfur species provides the opportunity for developing different transformations for the synthesis of interesting organic compounds. This tutorial review focuses on recent examples of the transition metal-catalyzed C-C bond forming reactions using organo-sulfur species.

Synthesis and HIV-1 RT inhibitory action of novel (4/6-substituted benzo[d]thiazol -2-yl)thiazolidin-4-ones. Divergence from the non-competitive inhibition mechanism.

Reverse transcriptase (RT) inhibitors play a major role in the therapy of human immunodeficiency virus type 1 (HIV-1) infection. Although, many compounds are already used as anti-HIV drugs, research on development of novel inhibitors continues, since drug resistant strains appear because of prolonged therapy. In this paper, we present the synthesis and evaluation of HIV-1 RT inhibitory action of eighteen novel (4/6-halogen/MeO/EtO-substituted benzo[d]thiazol-2-yl)thiazolidin-4-ones. The two more active compounds (IC50 : 0.04 µM and 0.25 µM) exhibited better inhibitory action than the reference compound, nevirapine. Docking analysis supports a stable binding of the most active derivative to the allosteric centre of RT. Kinetic analysis of two of the most active compounds indicate an uncompetitive inhibition mode. This is a desired characteristic, since mutations that affect activity of traditional non-competitive NNRTIs may not affect activity of compounds of this series. Interestingly, the less active derivatives (IC50 > 40 µM) exhibit a competitive mode of action.

The oligonucleotide binding (OB)-fold domain of KREPA4 is essential for stable incorporation into editosomes.

Most mitochondrial mRNAs in trypanosomatid parasites require uridine insertion/deletion RNA editing, a process mediated by guide RNA (gRNA) and catalyzed by multi-protein complexes called editosomes. The six oligonucleotide/oligosaccharide binding (OB)-fold proteins (KREPA1-A6), are a part of the common core of editosomes. They form a network of interactions among themselves as well as with the insertion and deletion sub-complexes and are essential for the stability of the editosomes. KREPA4 and KREPA6 proteins bind gRNA in vitro and are known to interact directly in yeast two-hybrid analysis. In this study, using several approaches we show a minimal interaction surface of the KREPA4 protein that is required for this interaction. By screening a series of N- and C-terminally truncated KREPA4 fragments, we show that a predicted α-helix of KREPA4 OB-fold is required for its interaction with KREPA6. An antibody against the KREPA4 α-helix or mutations of this region can eliminate association with KREPA6; while a peptide fragment corresponding to the α-helix can independently interact with KREPA6, thereby supporting the identification of KREPA4-KREPA6 interface. We also show that the predicted OB-fold of KREPA4; independent of its interaction with gRNA, is responsible for the stable integration of KREPA4 in the editosomes, and editing complexes co-purified with the tagged OB-fold can catalyze RNA editing. Therefore, we conclude that while KREPA4 interacts with KREPA6 through the α-helix region of its OB-fold, the entire OB-fold is required for its integration in the functional editosome, through additional protein-protein interactions.

Physico-chemical quality of drinking water in villages of Primary Health Centre, Waghodia, Gujarat (India).

16 water samples were collected to study the physical and chemical quality of water of main source of drinking water in the villages of Primary Health Centre, Waghodia of Vadodara district of Gujarat. The values recommended by Indian Standard for Drinking Water (IS 10500:1991) were used for comparison of observed values. The study indicates that the contamination problem in these villages is not alarming at present, but Waghodia being industrial town, ground water quality may deteriorate with passage of time, which needs periodical monitoring. The study provides the local area baseline data which may be useful for the comparison of future study.