Tropomyosin's periods are quasi-equivalent for actin binding but have specific regulatory functions

Tropomyosin is a coiled coil that associates N-terminus to C-terminus to form a continuous strand along both sides of the actin filament and regulates its function. One long, high molecular weight tropomyosin molecule spans the length of seven actin subunits. In these forms there is a 7-fold periodicity in noninterface residues that have been proposed to correspond to seven quasi-equivalent actin binding sites. Interruption of the stable, canonical coiled coil by residues that destabilize the interhelical interface, such as Ala clusters, is required for actin binding. Previous studies have shown that the N-terminal half of period 5 (residues 165-188) is critical for actin binding and regulatory function and that both the surface "consensus" residues and the embedded, destabilizing Ala cluster are required for function. In the present work we test the hypothesis of quasi-equivalence of tropomyosin's periodic sites by replacing the proposed binding sites by substituting the crucial period 5 region with regions of period 1 or 2. Replacement mutants were designed to test the importance of the coincidence of the consensus residues and a destabilizing interface. The results show that generic (interface instability) and specific periodic surface residues are essential for function and that the periods tested (periods 1, 2, and 5) are quasi-equivalent for actin binding. However, regulatory functions are period-specific: periods 1 and 5 for binding to actin in the force-producing state and period 5 for Ca2+-dependent regulation with troponin.     

An interaction of inorganic arsenic exposure with body weight and composition on type 2 diabetes indicators in Diversity Outbred mice

Mammalian Genome - Type 2 diabetes (T2D) is a complex metabolic disorder with no cure and high morbidity. Exposure to inorganic arsenic (iAs), a ubiquitous environmental contaminant, is associated...     

Biophysical characterization of mycobacterial model membranes and their interaction with rifabutin: Towards lipid-guided drug screening in tuberculosis

Lipid structure critically dictates the molecular interactions of drugs with membranes influencing passive diffusion, drug partitioning and accumulation, thereby underpinning a lipid-composition specific interplay. Spurring selective passive drug diffusion and uptake through membranes is an obvious solution to combat growing antibiotic resistance with minimized toxicities. However, the spectrum of complex mycobacterial lipids and lack thereof of suitable membrane platforms limits the understanding of mechanisms underlying drug-membrane interactions in tuberculosis. Herein, we developed membrane scaffolds specific to mycobacterial outer membrane and demonstrate them as improvised research platforms for investigating anti-tubercular drug interactions. Combined spectroscopy and microscopy results reveal an enhanced partitioning of model drug Rifabutin in trehalose dimycolate-containing mycobacterial membrane systems. These effects are apportioned to specific changes in membrane structure, order and fluidity leading to enhanced drug interaction. These findings on the membrane biophysical consequences of drug interactions will offer valuable insights for guiding the design of more effective antibiotic drugs coupled with tuned toxicity profiles.     

Transcriptome Sequencing of Chickpea (Cicer arietinum L.) Genotypes for Identification of Drought-Responsive Genes Under Drought Stress Condition

Plant Molecular Biology Reporter - Chickpea (Cicer arietinum L.) is a pulse crop valued for its high protein content, grown in semi-arid tropics and Mediterranean regions. Its yield remains...     

Chromatin-Based Classification of Genetically Heterogeneous AMLs into Two Distinct Subtypes with Diverse Stemness Phenotypes

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Quantitative proteomic analysis of venom from Southern India common krait (Bungarus caeruleus) and identification of poorly immunogenic toxins by immune-profiling against commercial antivenom

Objectives: To study the venom proteome composition of Southern India (SI) Common Krait (Bungarus caeruleus) and immunological cross-reactivity between venom against commercial antivenom.

Methods: Proteomic analysis was done by nano LC-MS/MS and toxins were quantitated by label-free analysis. The immunological cross-reactivity of venom towards polyvalent antivenom (PAV) was assessed by ELISA, Immunoblotting, and immuno-chromatographic methods.

Results: A total of 57 enzymatic and non-enzymatic proteins belonging to 12 snake venom protein families were identified. The three finger toxins (3FTx) (48.3%) and phospholipase A₂ (PLA₂) (37.6%) represented the most abundant non-enzymatic and enzymatic proteins, respectively. β-bungarotoxin (12.9%), a presynaptic neurotoxin, was also identified. The venom proteome composition is well correlated with its enzymatic activities, reported pharmacological properties, and clinical manifestations of krait envenomation. Immuno-cross-reactivity studies demonstrated better recognition of high molecular weight proteins (>45 kDa) of this venom by PAVs compared to low molecular weight (<15 kDa) toxins such as PLA₂ and 3FTxs.

Conclusion: The poor recognition of <15 kDa mass SI B. caeruleus venom proteins is of grave concern for the successful treatment of krait envenomation. Therefore, emphasis should be given to improve the immunization protocols and/or supplement of antibodies raised specifically against the <15 kDa toxins of this venom.     

Characterization of Genomic Deletion Efficiency Mediated by Clustered Regularly Interspaced Palindromic Repeats (CRISPR)/Cas9 Nuclease System in Mammalian Cells

The clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 nuclease system has provided a powerful tool for genome engineering. Double strand breaks may trigger nonhomologous end joining repair, leading to frameshift mutations, or homology-directed repair using an extrachromosomal template. Alternatively, genomic deletions may be produced by a pair of double strand breaks. The efficiency of CRISPR/Cas9-mediated genomic deletions has not been systematically explored. Here, we present a methodology for the production of deletions in mammalian cells, ranging from 1.3 kb to greater than 1 Mb. We observed a high frequency of intended genomic deletions. Nondeleted alleles are nonetheless often edited with inversions or small insertion/deletions produced at CRISPR recognition sites. Deleted alleles also typically include small insertion/deletions at predicted deletion junctions. We retrieved cells with biallelic deletion at a frequency exceeding that of probabilistic expectation. We demonstrate an inverse relationship between deletion frequency and deletion size. This work suggests that CRISPR/Cas9 is a robust system to produce a spectrum of genomic deletions to allow investigation of genes and genetic elements.     

Assessment of clinical outcomes in breast cancer patients treated with taxanes: multi-analytical approach

Polymorphisms in genes encoding CYPs (Phase I) and ABCB1 (Phase III) enzymes may attribute to variability of efficacy of taxanes. The present study aims to find the influence of CYP and ABCB1 gene polymorphisms on taxanes based clinical outcomes. 132 breast cancer patients treated with taxanes based chemotherapy were genotyped for CYP3A4*1B, CYP3A5*3, CYP1B1*3, CYP2C8*3, ABCB1 1236C>T, 2677G>T/A and 3435C>T polymorphisms using PCR-RFLP. Associations of genetic variants with clinical outcomes in terms of response in 58 patients receiving neo-adjuvant chemotherapy (NACT), and chemo-toxicity in 132 patients were studied. Multifactor dimensionality reduction (MDR) analysis was performed to evaluate higher order gene–gene interactions with clinical outcomes. Pathological response to taxane based NACT was associated with GA genotype as well as A allele of CYP3A5*3 polymorphism (P_corr = 0.0465, P_corr = 0.0465). Similarly, association was found in dominant model of CYP3A5*3 polymorphism with responders (P_corr = 0.0465). Haplotype analysis further revealed A_CYP3A4–A_CYP3A5 haplotype to be significantly associated with responders (P_corr = 0.048). In assessing toxicity, significant association of variant (TT) genotype and T allele of ABCB1 2677G>T/A polymorphism, was found with ‘grade 1 or no leucopenia’ (P_corr = 0.0465, P_corr = 0.048). On evaluating higher order gene–gene interaction models by MDR analysis, CYP3A5*3; ABCB11236C>T and ABCB1 2677G>T/A; ABCB1 3435C>T and CYP1B1*3 showed significant association with treatment response, grade 2–4 anemia and dose delay/reduction due to neutropenia (P = 0.024, P = 0.004, P = 0.026), respectively. Multi-analytical approaches may provide a better assessment of pharmacogenetic based treatment outcomes in breast cancer patients treated with taxanes.