A non-proteolytic release mechanism for HMCES-DNA-protein crosslinks.

The conserved protein HMCES crosslinks to abasic (AP) sites in ssDNA to prevent strand scission and the formation of toxic dsDNA breaks during replication. Here, we report a non-proteolytic release mechanism for HMCES-DNA-protein crosslinks (DPCs), which is regulated by DNA context. In ssDNA and at ssDNA-dsDNA junctions, HMCES-DPCs are stable, which efficiently protects AP sites against spontaneous incisions or cleavage by APE1 endonuclease. In contrast, HMCES-DPCs are released in dsDNA, allowing APE1 to initiate downstream repair. Mechanistically, we show that release is governed by two components. First, a conserved glutamate residue, within HMCES' active site, catalyses reversal of the crosslink. Second, affinity to the underlying DNA structure determines whether HMCES re-crosslinks or dissociates. Our study reveals that the protective role of HMCES-DPCs involves their controlled release upon bypass by replication forks, which restricts DPC formation to a necessary minimum.

Conflict monitoring and adaptation to affective stimuli as a function of ageing.

The current study investigated whether preference for positive affect would be observed in the context of a higher order control process with increasing age given the premise of affective prioritization with ageing. The study examined how affect interacted with cognitive control mechanisms across young, middle-aged and older adults. Conflict monitoring and adaptation for affective stimuli was examined with a face-word Stroop task using happy and fearful facial expressions. The participants' task was to detect the emotional expression (Happy or Fear) of the face shown with a distractor word (Happy or Fear) written across the face. Reaction time and accuracy data was analysed to compare adaptation effect and Stroop interference as a function of age, valence and previous trial congruence. The results demonstrated a stronger adaptation effect for negative affect in young adults and for positive affect in middle-aged adults and older adults. These results can be explained in terms of the socio-emotional selectivity theory of affective bias in the elderly and the involvement of attentional control mechanisms. This study empirically demonstrates shifts in affective bias towards positive affect with ageing through the implicit recruitment of cognitive control.

Synthesis of 2-Chloro-3-amino indenone derivatives and their evaluation as inhibitors of DNA dealkylation repair.

DNA alkylation damage, emanating from the exposure to environmental alkylating agents or produced by certain endogenous metabolic processes, affects cell viability and genomic stability. Fe(II)/2-oxoglutarate-dependent dioxygenase enzymes, such as Escherichia coli AlkB, are involved in protecting DNA from alkylation damage. Inspired by the natural product indenone derivatives reported to inhibit this class of enzymes, and a set of 2-chloro-3-amino indenone derivatives was synthesized and screened for their inhibitory properties against AlkB. The synthesis of 2-chloro-3-amino indenone derivatives was achieved from 2,3-dichloro indenones through addition-elimination method using alkyl/aryl amines under catalyst-free conditions. Using an in vitro reconstituted DNA repair assay, we have identified a 2-chloro-3-amino indenone compound 3o to be an inhibitor of AlkB. We have determined the binding affinity, mode of interaction, and kinetic parameters of inhibition of 3o and tested its ability to sensitize cells to methyl methanesulfonate that mainly produce DNA alkylation damage. This study established the potential of indenone-derived compounds as inhibitors of Fe(II)/2-oxoglutarate-dependent dioxygenase AlkB.

Neurocognitive mechanisms of affective conflict adaptation: An event related fMRI study.

Control of conflict can be seen in reduced effects of conflict following incompatible trials known as conflict adaptation. Such control mechanisms have been shown to depend on emotional content present in stimuli, which could be a motivational force for control adjustments. We explored the neural mechanisms of the interaction between proactive control in terms of conflict adaptation effect and emotions through an event related fMRI study involving an emotional Stroop effect (facial expression-emotional word paradigm) involving happy and angry expressions. Conflict adaptation was measured in terms of the reduction in Stroop effect as a function of previous trial congruence and previous trial emotion. Participants responded to the facial expression while ignoring the distractor word written over the face. Behavioral results showed larger Stroop effect for angry faces compared to happy faces. Conflict adaptation effect was greater for angry faces and was also influenced by previous trial emotion. Both priming and adaptation effects were observed. Stroop effect was correlated with activations in dorsal anterior cingulate. Emotion effect was correlated with activations in amygdala, fusiform face area (FFA), and insula along with the expected hemispheric asymmetry for positive and negative emotions in left vs right FFA, respectively. Conflict adaptation effect was correlated with activations in amygdala. In addition, activations in striatum supported the three-way interaction between emotion, previous and current trial congruence. Activation in dorsal anterior cingulate was correlated only with the overall Stroop effect in the current trial. Activations in amygdala and striatum were also found with facial expressions. The results indicate that emotion specific processing areas themselves such as amygdala and striatum may self-regulate and contribute toward enhanced proactive control mechanisms for task-relevant emotional stimuli. These findings further confirm the strong relationship between cognition and affect in the context of conflict monitoring and adjustments in cognitive control.

Determinants of Outcome in Convulsive Status Epilepticus in Adults: An Ambispective Study from Central India.

BACKGROUND: The determinants of the outcome in adult convulsive status epilepticus(CSE), also the implication of the value of mean arterial blood pressure (MAP), and random blood sugar at admission on the outcome are not clear. OBJECTIVES: The objective of this study is to look for the determinants of unfavorable outcome in CSE. MATERIALS AND METHODS: Ambispectively gathered data from 55 patients, treated consecutively with identical protocol during January 2010-December 2016, were analyzed. The demographic and clinical variables were identified and correlated with outcome in each individual. RESULTS: There were 65.45% males and 34.55% females. Favorable outcome (conscious and discharged) was seen in 63.6%, unfavorable (death 14.5%, absent cortical functions 10.9%, and inability to wean-off anesthetic agents 10.9%). The parameters associated with unfavorable outcome were female gender (odds ratio [OR]: 1.45), MAP ≤80 mmHg (OR: 2.57), time to first medical attention >5 h (OR: 127.8), and time to control clinical seizures >3.5 h (OR: 7.87). Almost 44.2% of patients with SE severity score >2 had unfavorable outcome (sensitivity 75% and specificity 45.7%). New scoring system, the CSE outcome score (CSEOS, developed by combining the predictors associated with higher odds of poor outcome), predicted the poor outcome with the sensitivity and specificity of 90% and 54.29%, respectively. DISCUSSION AND CONCLUSION: Low MAP and delay of >3.5 h in treatment initiation or seizure control are the key determinants of poor outcome in CSE. With the incorporation of CSEOS, we believe that our findings can be helpful in the process of clinical decision-making and prognostication of patients with CSE.

Heteroexpression of Mycobacterium leprae hypothetical protein ML0190 provides protection against DNA-alkylating agent methyl methanesulfonate.

Repair of DNA alkylation damage is essential for maintaining genome integrity and Fe(II)/2-oxoglutarate(2OG)-dependent dioxygenase family of enzymes play crucial role in repairing some of the alkylation damages. Alkylation repair protein-B (AlkB) of Escherichia coli belongs to Fe(II)/2OG-dependent dioxygenase family and carries out DNA dealkylation repair. We report here identification of a hypothetical Mycobacterium leprae protein (accession no. ML0190) from the genomic database and show that this 615-bp open reading frame encodes a protein with sequence and structural similarity to Fe(II)/2OG-dependent dioxygenase AlkB. We identified mRNA transcript of this gene in the M. leprae infected clinical skin biopsy samples isolated from the leprosy patients. Heterologous expression of ML0190 in methyl methane sulfonate (MMS) sensitive and DNA repair deficient strain of Saccharomyces cerevisiae and Escherichia coli resulted in resistance to alkylating agent MM. The results of the present study imply that Mycobacterium leprae ML0190 is involved in protecting the bacterial genome from DNA alkylation damage.

New biomarkers in brain trauma.

Brain-specific biomolecules are being increasingly investigated as a viable alternative to the clinical scores and radiological features, on which we still rely upon for stratification, therapy and predicting outcome in traumatic brain injury (TBI). TBI generally leads to release of various chemical compound within the cerebrospinal fluid (CSF) or blood depending on the severity of injury, which were studied variedly in last decades. However, most of these compounds being non-specific to brain, their applicability was challenged further. This review encompasses the novel and promising biomarkers being studied in the present decade, with encouraging results in laboratory and animal or human models.

Escherichia coli AlkB interacts with single-stranded DNA binding protein SSB by an intrinsically disordered region of SSB.

Intrinsically disordered regions (IDRs) of proteins often regulate function through interactions with folded domains. Escherichia coli single-stranded DNA binding protein SSB binds and stabilizes single-stranded DNA (ssDNA). The N-terminal of SSB contains characteristic OB (oligonucleotide/oligosaccharide-binding) fold which binds ssDNA tightly but non-specifically. SSB also forms complexes with a large number proteins via the C-terminal interaction domain consisting mostly of acidic amino acid residues. The amino acid residues located between the OB-fold and C-terminal acidic domain are known to constitute an IDR and no functional significance has been attributed to this region. Although SSB is known to bind many DNA repair protein, it is not known whether it binds to DNA dealkylation repair protein AlkB. Here, we characterize AlkB SSB interaction and demonstrate that SSB binds to AlkB via the IDR. We have established that AlkB-SSB interaction by in vitro pull-down and yeast two-hybrid analysis. We mapped the site of contact to be the residues 152-169 of SSB. Unlike most of the SSB-binding proteins which utilize C-terminal acidic domain for interaction, IDR of SSB is necessary and sufficient for AlkB interaction.

Indenone derivatives as inhibitor of human DNA dealkylation repair enzyme AlkBH3.

The mammalian AlkB homologue-3 (AlkBH3) is a member of the dioxygenase family of enzymes that in humans is involved in DNA dealkylation repair. Because of its role in promoting tumor cell proliferation and metastasis of cancer, extensive efforts are being directed in developing selective inhibitors for AlkBH3. Here we report synthesis, screening and evaluation of panel of arylated indenone derivatives as new class of inhibitors of AlkBH3 DNA repair activity. An efficient synthesis of 2,3-diaryl indenones from 2,3-dibromo indenones was achieved via Suzuki-Miyaura cross-coupling. Using a robust quantitative assay, we have obtained an AlkBH3 inhibitor that display specific binding and competitive mode of inhibition against DNA substrate. Finally, we established that this compound could prevent the proliferation of lung cancer cell line and enhance sensitivity to DNA damaging alkylating agent.

Early and rapid detection of UCHL1 in the serum of brain-trauma patients: a novel gold nanoparticle-based method for diagnosing the severity of brain injury.

The clinical diagnosis of traumatic brain injury (TBI) is based on neurological examination and neuro-imaging tools such as CT scanning and MRI. However, neurological examination at times may be confounded by consumption of alcohol or drugs and neuroimaging facilities may not be available at all centers. Human ubiquitin C-terminal hydrolase (UCHL1) is a well-accepted serum biomarker for severe TBI and can be used to detect the severity of a head injury. A reliable, rapid, cost effective, bedside and easy to perform method for the detection of UCHL1 is a pre-requisite for wide clinical applications of UCHL1 as a TBI biomarker. We developed a rapid detection method for UCHL1 using surface plasmon resonance of gold nanoparticles with a limit of detection (LOD) of 0.5 ng mL-1. It has a sensitivity and specificity of 100% each and meets an analytical precision similar to that of conventional sandwich ELISA but can be performed rapidly. Using this method we successfully detected UCHL1 in a cohort of 66 patients with TBI and were reliably able to distinguish mild TBI from moderate to severe TBI.

Escherichia coli single-stranded DNA binding protein SSB promotes AlkB-mediated DNA dealkylation repair.

Repair of alkylation damage in DNA is essential for maintaining genome integrity. Escherichia coli (E.coli) protein AlkB removes various alkyl DNA adducts including N1-methyladenine (N1meA) and N3-methylcytosine (N3meC) by oxidative demethylation. Previous studies showed that AlkB preferentially removes N1meA and N3meC from single-stranded DNA (ssDNA). It can also remove N1meA and N3meC from double-stranded DNA by base-flipping. Notably, ssDNA produced during DNA replication and recombination, remains bound to E. coli single-stranded DNA binding protein SSB and it is not known whether AlkB can repair methyl adduct present in SSB-coated DNA. Here we have studied AlkB-mediated DNA repair using SSB-bound DNA as substrate. In vitro repair reaction revealed that AlkB could efficiently remove N3meC adducts inasmuch as DNA length is shorter than 20 nucleotides. However, when longer N3meC-containing oligonuleotides were used as the substrate, efficiency of AlkB catalyzed reaction was abated compared to SSB-bound DNA substrate of identical length. Truncated SSB containing only the DNA binding domain could also support the stimulation of AlkB activity, suggesting the importance of SSB-DNA interaction for AlkB function. Using 70-mer oligonucleotide containing single N3meC we demonstrate that SSB-AlkB interaction promotes faster repair of the methyl DNA adducts.

Proactive and reactive control depends on emotional valence: a Stroop study with emotional expressions and words.

We examined proactive and reactive control effects in the context of task-relevant happy, sad, and angry facial expressions on a face-word Stroop task. Participants identified the emotion expressed by a face that contained a congruent or incongruent emotional word (happy/sad/angry). Proactive control effects were measured in terms of the reduction in Stroop interference (difference between incongruent and congruent trials) as a function of previous trial emotion and previous trial congruence. Reactive control effects were measured in terms of the reduction in Stroop interference as a function of current trial emotion and previous trial congruence. Previous trial negative emotions exert greater influence on proactive control than the positive emotion. Sad faces in the previous trial resulted in greater reduction in the Stroop interference for happy faces in the current trial. However, current trial angry faces showed stronger adaptation effects compared to happy faces. Thus, both proactive and reactive control mechanisms are dependent on emotional valence of task-relevant stimuli.

RecA stimulates AlkB-mediated direct repair of DNA adducts.

The Escherichia coli AlkB protein is a 2-oxoglutarate/Fe(II)-dependent demethylase that repairs alkylated single stranded and double stranded DNA. Immunoaffinity chromatography coupled with mass spectrometry identified RecA, a key factor in homologous recombination, as an AlkB-associated protein. The interaction between AlkB and RecA was validated by yeast two-hybrid assay; size-exclusion chromatography and standard pull down experiment and was shown to be direct and mediated by the N-terminal domain of RecA. RecA binding results AlkB-RecA heterodimer formation and RecA-AlkB repairs alkylated DNA with higher efficiency than AlkB alone.

Breastfeeding practices in baby friendly hospitals of Indore.

An interview based cross sectional study was conducted in two of the designated Baby Friendly Hospitals of Indore in the year 2008. None of the hospitals were having a written breastfeeding policy, which is routinely communicated to all the health workers and no regular training regarding the Programme was being imparted. There is a need to develop a BFHI Monitoring System to ensure that the status is kept in check. Training regarding essential Criteria of BFHI should be there for all the staff.