Association of Serum Biomarkers with the Mortality of Trauma Victims in a Level -1 Trauma Care Centre of Eastern India.

OBJECTIVE: To determine correlation of important biochemical laboratory investigations in different trauma patients and their degree of injury severity and overall mortality association. METHODS: In this hospital based retrospective observational study, 238 trauma patients were divided into two groups. Group I with injury severity score (ISS)<16 and group II with ISS>16. Haemoglobin (Hb), international normalized ratio, serum creatinine, blood urea nitogen (BUN), serum electrolyte, serum uric acid and liver function parameters were recorded and statistically analyzed. RESULTS: Group II had statistically significant (p<0.0001) elevated levels for referral pulse rate, creatinine, BUN, liver enzymes and decreased level in Hb% and potassium level compared to Group I. Strong positive correlation only exists between BUN and severity score, moderate positive correlation exists between creatinine, aspartate transaminase, and alanine transaminase, alkaline phosphatase and severity score and negative correlation between potassium and severity score. In this study, higher odds of high BUN and creatinine and lower potassium to normal values are associated with bad outcome such as higher mortality in the population of high ISS (>16). CONCLUSION: The study establishes the absolute need of doing three laboratory parameters (serum creatinine, serum blood urea nitrogen and serum potassium) instead of doing laboratory tests battery at the time of trauma victims admission and predicting survival among injured patients in trauma population from Indian settings.

DNA polymerase λ promotes error-free replication through Watson-Crick impairing N1-methyl-deoxyadenosine adduct in conjunction with DNA polymerase ζ.

In a previous study, we showed that replication through the N1-methyl-deoxyadenosine (1-MeA) adduct in human cells is mediated via three different Polι/Polθ, Polη, and Polζ-dependent pathways. Based on biochemical studies with these Pols, in the Polι/Polθ pathway, we inferred a role for Polι in the insertion of a nucleotide (nt) opposite 1-MeA and of Polθ in extension of synthesis from the inserted nt; in the Polη pathway, we inferred that this Pol alone would replicate through 1-MeA; in the Polζ pathway, however, the Pol required for inserting an nt opposite 1-MeA had remained unidentified. In this study, we provide biochemical and genetic evidence for a role for Polλ in inserting the correct nt T opposite 1-MeA, from which Polζ would extend synthesis. The high proficiency of purified Polλ for inserting a T opposite 1-MeA implicates a role for Polλ-which normally uses W-C base pairing for DNA synthesis-in accommodating 1-MeA in a syn confirmation and forming a Hoogsteen base pair with T. The potential of Polλ to replicate through DNA lesions by Hoogsteen base pairing adds another novel aspect to Polλ's role in translesion synthesis in addition to its role as a scaffolding component of Polζ. We discuss how the action mechanisms of Polλ and Polζ could be restrained to inserting a T opposite 1-MeA and extending synthesis thereafter, respectively.

Structural basis for polymerase η-promoted resistance to the anticancer nucleoside analog cytarabine.

Cytarabine (AraC) is an essential chemotherapeutic for acute myeloid leukemia (AML) and resistance to this drug is a major cause of treatment failure. AraC is a nucleoside analog that differs from 2'-deoxycytidine only by the presence of an additional hydroxyl group at the C2' position of the 2'-deoxyribose. The active form of the drug AraC 5'-triphosphate (AraCTP) is utilized by human replicative DNA polymerases to insert AraC at the 3' terminus of a growing DNA chain. This impedes further primer extension and is a primary basis for the drug action. The Y-family translesion synthesis (TLS) DNA polymerase η (Polη) counteracts this barrier to DNA replication by efficient extension from AraC-terminated primers. Here, we provide high-resolution structures of human Polη with AraC incorporated at the 3'-primer terminus. We show that Polη can accommodate AraC at different stages of the catalytic cycle, and that it can manipulate the conformation of the AraC sugar via specific hydrogen bonding and stacking interactions. Taken together, the structures provide a basis for the ability of Polη to extend DNA synthesis from AraC terminated primers.

Mechanism of error-free DNA synthesis across N1-methyl-deoxyadenosine by human DNA polymerase-ι.

N1-methyl-deoxyadenosine (1-MeA) is formed by methylation of deoxyadenosine at the N1 atom. 1-MeA presents a block to replicative DNA polymerases due to its inability to participate in Watson-Crick (W-C) base pairing. Here we determine how human DNA polymerase-ι (Polι) promotes error-free replication across 1-MeA. Steady state kinetic analyses indicate that Polι is ~100 fold more efficient in incorporating the correct nucleotide T versus the incorrect nucleotide C opposite 1-MeA. To understand the basis of this selectivity, we determined ternary structures of Polι bound to template 1-MeA and incoming dTTP or dCTP. In both structures, template 1-MeA rotates to the syn conformation but pairs differently with dTTP versus dCTP. Thus, whereas dTTP partakes in stable Hoogsteen base pairing with 1-MeA, dCTP fails to gain a "foothold" and is largely disordered. Together, our kinetic and structural studies show how Polι maintains discrimination between correct and incorrect incoming nucleotide opposite 1-MeA in preserving genome integrity.

The architecture of yeast DNA polymerase ζ.

DNA polymerase ζ (Polζ) is specialized for the extension step of translesion DNA synthesis (TLS). Despite its central role in maintaining genome integrity, little is known about its overall architecture. Initially identified as a heterodimer of the catalytic subunit Rev3 and the accessory subunit Rev7, yeast Polζ has recently been shown to form a stable four-subunit enzyme (Polζ-d) upon the incorporation of Pol31 and Pol32, the accessory subunits of yeast Polδ. To understand the 3D architecture and assembly of Polζ and Polζ-d, we employed electron microscopy. We show here how the catalytic and accessory subunits of Polζ and Polζ-d are organized relative to each other. In particular, we show that Polζ-d has a bilobal architecture resembling the replicative polymerases and that Pol32 lies in proximity to Rev7. Collectively, our study provides views of Polζ and Polζ-d and a structural framework for understanding their roles in DNA damage bypass.

Rates of intercalator-driven platination of DNA determined by a restriction enzyme cleavage inhibition assay.

A restriction enzyme cleavage inhibition assay was designed to determine the rates of DNA platination by four non-cross-linking platinum-acridine agents represented by the formula [Pt(am(2))LCl](NO(3))(2), where am is a diamine nonleaving group and L is an acridine derived from the intercalator 1-[2-(acridin-9-ylamino)ethyl]-1,3-dimethylthiourea (ACRAMTU). The formation of monofunctional adducts in the target sequence 5'-CGA was studied in a 40-base-pair probe containing the EcoRI restriction site GAATTC. The time dependence of endonuclease inhibition was quantitatively analyzed by polyacrylamide gel electrophoresis. The formation of monoadducts is approximately 3 times faster with double-stranded DNA than with simple nucleic acid fragments. Compound 1 (am(2) is ethane-1,2-diamine, L is ACRAMTU) reacts with a first-order rate constant of k (obs) = 1.4 ± 0.37 × 10(-4) s(-1) (t (1/2) = 83 ± 22 min). Replacement of the thiourea group in ACRAMTU with an amidine group (compound 2) accelerates the rate by fourfold (k (obs) = 5.7 ± 0.58 × 10(-4) s(-1), t (1/2) = 21 ± 2 min), and introduction of a propane-1,3-diamine nonleaving group results in a 1.5-fold enhancement in reactivity (compound 3, k (obs) = 2.1 ± 0.40 × 10(-4) s(-1), t (1/2) = 55 ± 10 min) compared with the prototype. Derivative 4, containing a 4,9-disubstituted acridine threading intercalator, was the least reactive compound in the series (k (obs) = 1.1 ± 0.40 × 10(-4) s(-1), t (1/2) = 104 ± 38 min). The data suggest a correlation may exist between the binding rates and the biological activity of the compounds. Potential pharmacological advantages of rapid formation of cytotoxic monofunctional adducts over the common purine-purine cross-links are discussed.

A non-cross-linking platinum-acridine agent with potent activity in non-small-cell lung cancer.

The cytotoxic complex, [PtCl(Am)2(ACRAMTU)](NO3)2 (1) ((Am)2 = ethane-1,2-diamine, en; ACRAMTU = 1-[2-(acridin-9-ylamino)ethyl]-1,3-dimethylthiourea), is a dual platinating/intercalating DNA binder that, unlike clinical platinum agents, does not induce DNA cross-links. Here, we demonstrate that substitution of the thiourea with an amidine group leads to greatly enhanced cytotoxicity in H460 non-small-cell lung cancer (NSCLC) in vitro and in vivo. Two complexes were synthesized: 4a (Am2 = en) and 4b (Am = NH3), in which N-[2-(acridin-9-ylamino)ethyl]-N-methylpropionamidine replaces ACRAMTU. Complex 4a proves to be a more efficient DNA binder than complex 1 and induces adducts in sequences not targeted by the prototype. Complexes 4a and 4b induce H460 cell kill with IC(50) values of 28 and 26 nM, respectively, and 4b slows tumor growth in a H460 mouse xenograft study by 40% when administered at a dose of 0.5 mg/kg. Compound 4b is the first non-cross-linking platinum agent endowed with promising activity in NSCLC.

Tuning the DNA conformational perturbations induced by cytotoxic platinum-acridine bisintercalators: effect of metal cis/trans isomerism and DNA threading groups.

Four highly charged, water soluble platinum-acridine bisintercalating agents have been synthesized. Depending on the cis/trans isomerism of the metal and the nature of the acridine side chains, bisintercalation induces/stabilizes the classical Watson-Crick B-form or a non-B-form. Circular dichroism spectra and chemical footprinting experiments suggest that 4, the most active derivative in HL-60 cells, produces a structurally severely perturbed DNA with features of a Hoogsteen base-paired biopolymer.

Effect of the diamine nonleaving group in platinum-acridinylthiourea conjugates on DNA damage and cytotoxicity.

The following complexes of type [PtCl(R)(ACRAMTU)](NO3)2 (ACRAMTU = 1-[2-(acridin-9-ylamino)ethyl]-1,3-dimethylthiourea)), derived from prototype 1 (with R = ethane-1,2-diamine), were synthesized: 2 (with R = (1R,2R)-1,2-diaminocyclohexane), 3 (with R = propane-1,3-diamine), 4 (with R = N1,N1,N2,N2-tetramethylethane-1,2-diamine), and 5 (with R = 2,2'-bipyridine). The DNA sequence specificity of the conjugates and their antiproliferative potential in HL-60 and H460 cells were investigated. Conjugate 3 showed the strongest non-cisplatin-type DNA damage in polymerase stop assays and superior cell kill efficacy in H460 lung cancer (IC50 = 70 nM).

Characterization of the bisintercalative DNA binding mode of a bifunctional platinum-acridine agent.

The DNA interactions of PT-BIS(ACRAMTU) ([Pt(en)(ACRAMTU)2](NO3)4; ACRAMTU = 1-[2-(acridin-9-ylamino)ethyl]-1,3-dimethylthiourea, en = ethylenediamine), a bifunctional platinum-acridine conjugate, have been studied in native and synthetic double-stranded DNAs and model duplexes using various biophysical techniques. These include ethidium-DNA fluorescence quenching and thermal melting experiments, circular dichroism (CD) spectroscopy and plasmid unwinding assays. In addition, the binding mode was studied in a short octamer by NMR spectroscopy in conjunction with molecular modeling. In alternating copolymers, PT-BIS(ACRAMTU) shows a distinct preference for poly(dA-dT)2, which is approximately 3-fold higher than that of ACRAMTU. In the ligand-oligomer complex, d(GCTATAGC)2.PT-BIS(ACRAMTU) (complex I*), PT-BIS(ACRAMTU) increases the thermal stability of the B-form host duplex by DeltaT(m) > 30 K (CD and UV melting experiments). The agent unwinds pSP73 plasmid DNA by 44(+/-2) degrees per bound molecule, indicating bisintercalative binding. A 2-D NMR study unequivocally demonstrates that PT-BIS(ACRAMTU)'s chromophores deeply bisintercalate into the 5'-TA/TA base pair steps in I*, while the platinum linker lies in the minor groove. An AMBER model reflecting the NMR results shows that bracketing of the central AT base pairs in a classical nearest neighbor excluded fashion is feasible. PT-BIS(ACRAMTU) inhibits DNA hydrolysis by BstZ17 I at the enzyme's restriction site, GTA downward arrowTAC. Possible consequences for other relevant DNA-protein interactions, such as those involved in TATA-box-mediated transcription initiation and the utility of the platinum-intercalator technology for the design of sequence-specific agents are discussed.