An Aurora B-RPA signaling axis secures chromosome segregation fidelity.

Errors in chromosome segregation underlie genomic instability associated with cancers. Resolution of replication and recombination intermediates and protection of vulnerable single-stranded DNA (ssDNA) intermediates during mitotic progression requires the ssDNA binding protein Replication Protein A (RPA). However, the mechanisms that regulate RPA specifically during unperturbed mitotic progression are poorly resolved. RPA is a heterotrimer composed of RPA70, RPA32 and RPA14 subunits and is predominantly regulated through hyperphosphorylation of RPA32 in response to DNA damage. Here, we have uncovered a mitosis-specific regulation of RPA by Aurora B kinase. Aurora B phosphorylates Ser-384 in the DNA binding domain B of the large RPA70 subunit and highlights a mode of regulation distinct from RPA32. Disruption of Ser-384 phosphorylation in RPA70 leads to defects in chromosome segregation with loss of viability and a feedback modulation of Aurora B activity. Phosphorylation at Ser-384 remodels the protein interaction domains of RPA. Furthermore, phosphorylation impairs RPA binding to DSS1 that likely suppresses homologous recombination during mitosis by preventing recruitment of DSS1-BRCA2 to exposed ssDNA. We showcase a critical Aurora B-RPA signaling axis in mitosis that is essential for maintaining genomic integrity.

Site-specific labeling of SBDS to monitor interactions with the 60S ribosomal subunit.

The Shwachman-Diamond syndrome (SDS) is a rare inherited ribosomopathy that is predominantly caused by mutations in the Shwachman-Bodian-Diamond Syndrome gene (SBDS). SBDS is a ribosomal maturation factor that is essential for the release of eukaryotic translation initiation factor 6 (eIF6) from 60S ribosomal subunits during the late stages of 60S maturation. Release of eIF6 is critical to permit inter-subunit interactions between the 60S and 40S subunits and to form translationally competent 80S monosomes. SBDS has three key domains that are highly flexible and adopt varied conformations in solution. To better understand the domain dynamics of SBDS upon binding to 60S and to assess the effects of SDS-disease specific mutations, we aimed to site-specifically label individual domains of SBDS. Here we detail the generation of a fluorescently labeled SBDS to monitor the dynamics of select domains upon binding to 60S. We describe the incorporation of 4-azido-l-phenylalanine (4AZP), a noncanonical amino acid in human SBDS. Site-specific labeling of SBDS using fluorophore and assessment of 60S binding activity are also described. Such labeling approaches to capture the interactions of individual domains of SBDS with 60S are also applicable to study the dynamics of other multi-domain proteins that interact with the ribosomal subunits.

Dynamic states of eIF6 and SDS variants modulate interactions with uL14 of the 60S ribosomal subunit.

Assembly of ribosomal subunits into active ribosomal complexes is integral to protein synthesis. Release of eIF6 from the 60S ribosomal subunit primes 60S to associate with the 40S subunit and engage in translation. The dynamics of eIF6 interaction with the uL14 (RPL23) interface of 60S and its perturbation by somatic mutations acquired in Shwachman-Diamond Syndrome (SDS) is yet to be clearly understood. Here, by using a modified strategy to obtain high yields of recombinant human eIF6 we have uncovered the critical interface entailing eight key residues in the C-tail of uL14 that is essential for physical interactions between 60S and eIF6. Disruption of the complementary binding interface by conformational changes in eIF6 disease variants provide a mechanism for weakened interactions of variants with the 60S. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) analyses uncovered dynamic configurational rearrangements in eIF6 induced by binding to uL14 and exposed an allosteric interface regulated by the C-tail of eIF6. Disrupting key residues in the eIF6-60S binding interface markedly limits proliferation of cancer cells, which highlights the significance of therapeutically targeting this interface. Establishing these key interfaces thus provide a therapeutic framework for targeting eIF6 in cancers and SDS.

New records of elasmobranchs in the Bay of Bengal, Bangladesh: further taxonomic research is essential.

To evaluate the species diversity and strengthen the taxonomic identification of elasmobranchs in the Bay of Bengal, Bangladesh, a study was conducted in the southeast coastal region between January 2016 and March 2018. Using morphological and genetic identification techniques, this study presents 22 species from the region. Thirteen of these are new records. The new records consist of eight species from the family Dasyatidae, and one each from Mobulidae, Rhinobatidae, Narcinidae, Hemiscylliidae and Triakidae. Furthermore, four occurrences are first verified reports, and five are potential new records requiring further taxonomic investigation.

Stimulatory effect of Holy basil and Thai basil on mouse spleen cell proliferation.

Study was conducted on mouse spleen cells, cultured and incubated in-vitro with Holy basil and Thai basil, to observe their effect on proliferation. Four dilutions, namely 1:1, 1:5, 1:25, and 1:125, for both Holy basil and Thai Basil were used separately, in presence and absence of mitogen, Concanavalin A (Con A) to stimulate the T cells. Cell proliferation was monitored by 3 H- thymidine radioisotope incorporation. Spleen cells (macrophages, B and T cells) showed significantly more proliferation at 1:1 dilution than control (cells with no factor), incubated with Holy basil (in assay without Con A). Spleen T cells, however, did not show any significance in proliferation at same dilution, 1:1, with Holy basil with Con A. All other dilutions (with or without Con A), for either Holy basil or Thai basil, did not show any significant changes in proliferation when compared to control.

DNA analysis of elasmobranch products originating from Bangladesh reveals unregulated elasmobranch fishery and trade on species of global conservation concern.

Trade involving elasmobranch products in Bangladesh is a four-decade-long practice in large scale and there is little understanding of its impact on species composition, population, and subsequent conservation. Capacity for monitoring and identification is lacking in landing and shark processing centres. A rapid survey and collection of tissue samples were performed in three landings and nine shark processing centres between 2016 and 2017 in the south-eastern coastal region of Bangladesh. Sequencing for a 707-bp fragment of the mitochondrial cytochrome c oxidase subunit I (COI) gene was used to assess the taxonomic status and species composition from 71 elasmobranch tissue samples collected from the shark processing centre only. Good quality COI sequences were obtained for 34 specimens representing 21 species-the majority of which are threatened with extinction. A total of ten species of sharks (Carcharhinus brevipinna, C. amboinensis, C. leucas, C. sorrah, C. amblyrhynchoides, Chiloscyllium burmensis, Galeocerdo cuvier, Rhincodon typus, Scoliodon laticaudus, and Sphyrna lewini), eleven species of rays (Aetomylaeus maculatus, Gymnura poecilura, Mobula mobular, M. kuhlii, Neotrygon indica, Pateobatis uarnacoides, Rhinoptera javanica, and R. jayakari), including three species of guitarfish (Glaucostegus granulatus, G. obtusus, and G. typus), were identified. Four species (14.7% of samples) were found to be listed in the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) in Appendix II. Sixteen species (59% of the specimens) were threatened with extinction according to IUCN Red List, whereas 41% were data deficient or not assessed. The results have important implications for the management of regional fisheries and the conservation of elasmobranchs as they 1) represent a preliminary understanding of elasmobranch diversity in trade; 2) depict a lack of awareness and monitoring; and 3) demonstrate a need for urgent monitoring and regulation of elasmobranch trade in Bangladesh.

Age-Specific Cut-off Values of Amino Acids and Acylcarnitines for Diagnosis of Inborn Errors of Metabolism Using Liquid Chromatography Tandem Mass Spectrometry.

Liquid Chromatography tandem mass spectrometry (LC-MS/MS) is used for the diagnosis of more than 30 inborn errors of metabolisms (IEMs). Accurate and reliable diagnosis of IEMs by quantifying amino acids (AAs) and acylcarnitines (ACs) using LC-MS/MS systems depend on the establishment of age-specific cut-offs of the analytes. This study aimed to (1) determine the age-specific cut-off values of AAs and ACs in Bangladesh and (2) validate the LC-MS/MS method for diagnosis of the patients with IEMs. A total of 570 enrolled healthy participants were divided into 3 age groups, namely, (1) newborns (1-7 days), (2) 8 days-7 years, and (3) 8-17 years, to establish the age-specific cut-offs for AAs and ACs. Also, 273 suspected patients with IEMs were enrolled to evaluate the reliability of the established cut-off values. Quantitation of AAs and ACs was performed on an automated LC-MS/MS system using dried blood spot (DBS) cards. Then the specimens of the enrolled clinically suspected patients were analyzed by the established method. Nine patients came out as screening positive for different IEMs, including two borderline positive cases of medium-chain acyl-CoA dehydrogenase deficiency (MCAD). A second-tier test for confirmation of the screening positive cases was conducted by urinary metabolic profiling using gas chromatography- mass spectrometry (GC-MS). Out of 9 cases that came out as screening positive by LC-MS/MS, seven cases were confirmed by urinary GC-MS analysis including 3 cases with phenylketonuria, 1 with citrullinemia type II, 1 with methylmalonic acidemia, 1 with isovaleric acidemia and 1 with carnitine uptake defect. Two borderline positive cases with MCAD were found negative by urinary GC-MS analysis. In conclusion, along with establishment of a validated LC-MS/MS method for quantitation of AAs and ACs from the DBS cards, the study also demonstrates the presence of predominantly available IEMs in Bangladesh.

High resolution melting curve analysis targeting the HBB gene mutational hot-spot offers a reliable screening approach for all common as well as most of the rare beta-globin gene mutations in Bangladesh.

BACKGROUND: Bangladesh lies in the global thalassemia belt, which has a defined mutational hot-spot in the beta-globin gene. The high carrier frequencies of beta-thalassemia trait and hemoglobin E-trait in Bangladesh necessitate a reliable DNA-based carrier screening approach that could supplement the use of hematological and electrophoretic indices to overcome the barriers of carrier screening. With this view in mind, the study aimed to establish a high resolution melting (HRM) curve-based rapid and reliable mutation screening method targeting the mutational hot-spot of South Asian and Southeast Asian countries that encompasses exon-1 (c.1 - c.92), intron-1 (c.92 + 1 - c.92 + 130) and a portion of exon-2 (c.93 - c.217) of the HBB gene which harbors more than 95% of mutant alleles responsible for beta-thalassemia in Bangladesh. RESULTS: Our HRM approach could successfully differentiate ten beta-globin gene mutations, namely c.79G > A, c.92 + 5G > C, c.126_129delCTTT, c.27_28insG, c.46delT, c.47G > A, c.92G > C, c.92 + 130G > C, c.126delC and c.135delC in heterozygous states from the wild type alleles, implying the significance of the approach for carrier screening as the first three of these mutations account for ~85% of total mutant alleles in Bangladesh. Moreover, different combinations of compound heterozygous mutations were found to generate melt curves that were distinct from the wild type alleles and from one another. Based on the findings, sixteen reference samples were run in parallel to 41 unknown specimens to perform direct genotyping of the beta-thalassemia specimens using HRM. The HRM-based genotyping of the unknown specimens showed 100% consistency with the sequencing result. CONCLUSIONS: Targeting the mutational hot-spot, the HRM approach could be successfully applied for screening of beta-thalassemia carriers in Bangladesh as well as in other countries of South Asia and Southeast Asia. The approach could be a useful supplement of hematological and electrophortic indices in order to avoid false positive and false negative results.