Engineering Nucleotide Specificity of Succinyl-CoA Synthetase in Blastocystis: The Emerging Role of Gatekeeper Residues.

PublishedJournal ArticleThis is the final version of the article. Available from American Chemical Society via the DOI in this record.Charged, solvent-exposed residues at the entrance to the substrate binding site (gatekeeper residues) produce electrostatic dipole interactions with approaching substrates, and control their access by a novel mechanism called "electrostatic gatekeeper effect". This proof-of-concept study demonstrates that the nucleotide specificity can be engineered by altering the electrostatic properties of the gatekeeper residues outside the binding site. Using Blastocystis succinyl-CoA synthetase (SCS, EC 6.2.1.5), we demonstrated that the gatekeeper mutant (ED) resulted in ATP-specific SCS to show high GTP specificity. Moreover, nucleotide binding site mutant (LF) had no effect on GTP specificity and remained ATP-specific. However, via combination of the gatekeeper mutant with the nucleotide binding site mutant (ED+LF), a complete reversal of nucleotide specificity was obtained with GTP, but no detectable activity was obtained with ATP. This striking result of the combined mutant (ED+LF) was due to two changes; negatively charged gatekeeper residues (ED) favored GTP access, and nucleotide binding site residues (LF) altered ATP binding, which was consistent with the hypothesis of the "electrostatic gatekeeper effect". These results were further supported by molecular modeling and simulation studies. Hence, it is imperative to extend the strategy of the gatekeeper effect in a different range of crucial enzymes (synthetases, kinases, and transferases) to engineer substrate specificity for various industrial applications and substrate-based drug design.Work is supported by the National Institute of Malaria Research, Indian Council of Medical Research, New Delhi and Dept. of Biotechnology, New Delhi. K.C.P. is a recipient of the Prof. Ramalingaswami Fellowship (Department of Biotechnology, Government of India (BT/HRD/35/02/2006), K.V. is a recipient of UGC Senior Research Fellowship, M.v.d.G. is grateful for support from the University of Exeter and the Wellcome Trust (078566/A/05/Z)

Primary sclerosing cholangitis in India

Six patients with PSC have been diagnosed and followed up at a centre in Northern India for periods upto 4 years. They all presented with cholestatic jaundice and cholangitis, but one of them subsequently presented with variceal haemorrhage. Cholangiographic features were the most characteristic and included irregular narrowing and segmental dilatation of the biliary radicles giving them a beaded appearance. Treating them was most frustrating. Two of them died of hepatic encephalopathy, others have also continued to be sick during the follow-up

Chronic calcific pancreatitis: clinical profile in northern India

Twenty three patients with chronic calcific pancreatitis of the tropics in Northern India were prospectively studied. All had pancreatic calcification and ERCP changes typical of chronic pancreatitis, the most predominant being ductal dilatation which was detected in all patients by both ERCP and by ultrasonography. Pain was present in 19 (83%) patients and diabetes in 11 (48%) patients. Exocrine pancreatic dysfunction was uncommon, steatorrhoea being present in only 9% of patients. Ten of the 11 patients with diabetes required insulin for control and one case was able to be controlled by an oral antidiabetic agent. Two patients developed ketoacidosis during acute episodes of pancreatitis, 3 patients had peripheral neuropathy and one patient had visual changes. Recurrent severe pain was the reason for operation in 7 patients. All had a lateral pancreaticojejunostomy. In order to obtain an objective assessment of pain, a scoring system was developed to grade its severity according to its intensity, frequency and consequences. Six patients who preoperatively had a pain score of 15 or more (out of a maximum score of 24) attained significant relief after the surgery. We feel this scoring system may provide an easy objective assessment of pain in the subsequent follow-up of these patients

Estimating Tourist Growth at Destination Sites: A Mathematical Equation and a Connectivity Model Through Mobile Application

لقد سعى باحثو العلوم الاجتماعية إلى عرض الأدبيات المختلفة لأنظمة التوصية المتنقلة الفردية لمساعدة أصحاب المصلحة في قطاع السياحة. إن التحدي المتمثل في صياغة معادلة رياضية للتنبؤ بالنمو السياحي في مواقع محددة ينشأ بسبب الظروف البيئية المتنوعة الفريدة لكل وجهة. يسعى هذا البحث إلى تطوير نموذج لتطبيقات الهاتف المحمول يعزز الاتصال بين أصحاب المصلحة الرئيسيين في مجال السياحة. داخل التطبيق، يمكن لأصحاب المصلحة استرداد المعلومات حول الآخرين بسهولة والتفاعل مباشرة مع السلطات ذات الصلة دون الخروج من التطبيق. يوفر التطبيق أيضًا إرشادات ملاحية للوجهات السياحية. وتستكشف الدراسة معدل النمو النوعي للسياح، بافتراض تضاعف أعداد السياح بعد فترة زمنية محددة. في حين أنه قد يكون هناك نمو هائل مبدئي في أعداد السياح في الوجهة، إلا أن العدد يستقر في النهاية. تم استخدام معادلة مونود مع المعادلة السياحية للحصول على تمثيل شامل في هذه الدراسة. بالإضافة إلى ذلك، يتعمق البحث في التحليل الرسومي لشروط الجدوى التي يقترحها نموذج الاتصال الخاص بـCasagrand i و Rinaldi. و من الضروري ملاحظة أن هذا التحليل يظل مجردًا، وأن إنشاء نموذج رياضي رياضي قابل للتطبيق عالميًا لكل وجهة يكاد يكون مستحيلًا.Social science researchers have endeavored with the literature showcasing various individual mobile recommendation systems to assist stakeholders in the tourism sector. The challenge of formulating a mathematical equation to predict tourist growth at specific sites arises due to the diverse environmental conditions unique to each destination. This research seeks to develop a mobile application model fostering connectivity among significant tourism stakeholders. Within the app, stakeholders can effortlessly retrieve information about others and directly engage with relevant authorities without exiting the application. The app also provides navigation guidance to tourist destinations. The study explores the specific growth rate of tourists, assuming a doubling of tourist numbers after a specific time interval has been illustrated. While there may be an initial exponential growth in tourist numbers at a destination, the count eventually stabilizes. The Monod equation is employed in conjunction with the tourist equation for a comprehensive representation in this study. Additionally, the research delves into the graphical analysis of the feasibility conditions proposed by Casagrandi and Rinaldi's connectivity model. It is essential to note that this analysis remains abstract, and the creation of a universally applicable mathematical tourism model for every destination proves nearly impossible

Characterization of an unusual bipolar helicase encoded by bacteriophage T5

Bacteriophage T5 has a 120 kb double-stranded linear DNA genome encoding most of the genes required for its own replication. This lytic bacteriophage has a burst size of ∼500 new phage particles per infected cell, demonstrating that it is able to turn each infected bacterium into a highly efficient DNA manufacturing machine. To begin to understand DNA replication in this prodigious bacteriophage, we have characterized a putative helicase encoded by gene D2. We show that bacteriophage T5 D2 protein is the first viral helicase to be described with bipolar DNA unwinding activities that require the same core catalytic residues for unwinding in either direction. However, unwinding of partially single- and double-stranded DNA test substrates in the 3′–5′ direction is more robust and can be distinguished from the 5′–3′ activity by a number of features including helicase complex stability, salt sensitivity and the length of single-stranded DNA overhang required for initiation of helicase action. The presence of D2 in an early gene cluster, the identification of a putative helix-turn-helix DNA-binding motif outside the helicase core and homology with known eukaryotic and prokaryotic replication initiators suggest an involvement for this unusual helicase in DNA replication initiation

Caenorhabditis elegans ALG-1 antimorphic mutations uncover functions for Argonaute in microRNA guide strand selection and passenger strand disposal

MicroRNAs are regulators of gene expression whose functions are critical for normal development and physiology. We have previously characterized mutations in a Caenorhabditis elegans microRNA-specific Argonaute ALG-1 (Argonaute-like gene) that are antimorphic [alg-1(anti)]. alg-1(anti) mutants have dramatically stronger microRNA-related phenotypes than animals with a complete loss of ALG-1. ALG-1(anti) miRISC (microRNA induced silencing complex) fails to undergo a functional transition from microRNA processing to target repression. To better understand this transition, we characterized the small RNA and protein populations associated with ALG-1(anti) complexes in vivo. We extensively characterized proteins associated with wild-type and mutant ALG-1 and found that the mutant ALG-1(anti) protein fails to interact with numerous miRISC cofactors, including proteins known to be necessary for target repression. In addition, alg-1(anti) mutants dramatically overaccumulated microRNA* (passenger) strands, and immunoprecipitated ALG-1(anti) complexes contained nonstoichiometric yields of mature microRNA and microRNA* strands, with some microRNA* strands present in the ALG-1(anti) Argonaute far in excess of the corresponding mature microRNAs. We show complex and microRNA-specific defects in microRNA strand selection and microRNA* strand disposal. For certain microRNAs (for example mir-58), microRNA guide strand selection by ALG-1(anti) appeared normal, but microRNA* strand release was inefficient. For other microRNAs (such as mir-2), both the microRNA and microRNA* strands were selected as guide by ALG-1(anti), indicating a defect in normal specificity of the strand choice. Our results suggest that wild-type ALG-1 complexes recognize structural features of particular microRNAs in the context of conducting the strand selection and microRNA* ejection steps of miRISC maturation

Guiding the Design of Synthetic DNA-Binding Molecules with Massively Parallel Sequencing

Genomic applications of DNA-binding molecules require an unbiased knowledge of their high affinity sites. We report the high-throughput analysis of pyrrole-imidazole polyamide DNA-binding specificity in a 10^(12)-member DNA sequence library using affinity purification coupled with massively parallel sequencing. We find that even within this broad context, the canonical pairing rules are remarkably predictive of polyamide DNA-binding specificity. However, this approach also allows identification of unanticipated high affinity DNA-binding sites in the reverse orientation for polyamides containing β/Im pairs. These insights allow the redesign of hairpin polyamides with different turn units capable of distinguishing 5′-WCGCGW-3′ from 5′-WGCGCW-3′. Overall, this study displays the power of high-throughput methods to aid the optimal targeting of sequence-specific minor groove binding molecules, an essential underpinning for biological and nanotechnological applications

Thermophysical Properties of Binary Liquid Systems of Ethanoic Acid, Propanoic Acid, and Butanoic Acid with Benzene or Acetophenone

The density (), viscosity (), and surface tension () of binary mixtures of carboxylic acids (CAs) (ethanoic acid (EA), propanoic acid (PA), butanoic acid (BA)) + benzene (BEN) or acetophenone (ACT) have been measured at 298.15, 308.15, and 318.15 K. From the experimental results, excess values of molar volume (), viscosity (), Gibb's free energy for the activation of low (), and surface tension () were evaluated and fitted to a Redlich-Kister type of equation. The parameter “” of Grunberg and Nissan expression has also been calculated. From the sign and magnitude of , , , , and “” values, it is concluded that specific interactions are present in CA+ACT system and these interactions are absent in CA + BEN mixtures. Various viscosity and surface tension models have been used to test the consistency of the data.</jats:p

A Novel Histone Deacetylase Complex in the Control of Transcription and Genome Stability

The acetylation state of histones, controlled by histone acetyltransferases (HATs) and deacetylases (HDACs), profoundly affects DNA transcription and repair by modulating chromatin accessibility to the cellular machinery. The Schizosaccharomyces pombe HDAC Clr6 (human HDAC1) binds to different sets of proteins that define functionally distinct complexes: I, I', and II. Here, we determine the composition, architecture, and functions of a new Clr6 HDAC complex, I'', delineated by the novel proteins Nts1, Mug165, and Png3. Deletion of nts1 causes increased sensitivity to genotoxins and deregulated expression of Tf2 elements, long noncoding RNA, and subtelomeric and stress-related genes. Similar, but more pervasive, phenotypes are observed upon Clr6 inactivation, supporting the designation of complex I'' as a mediator of a key subset of Clr6 functions. We also reveal that with the exception of Tf2 elements, the genome-wide loading sites and loci regulated by Clr6 I″ do not correlate. Instead, Nts1 loads at genes that are expressed in midmeiosis, following oxidative stress, or are periodically expressed. Collective data suggest that Clr6 I'' has (i) indirect effects on gene expression, conceivably by mediating higher-order chromatin organization of subtelomeres and Tf2 elements, and (ii) direct effects on the transcription of specific genes in response to certain cellular or environmental stimuli

Porcupine Inhibition Disrupts Mitochondrial Function and Homeostasis in WNT Ligand-Addicted Pancreatic Cancer.

WNT signaling promotes pancreatic ductal adenocarcinoma (PDAC) through diverse effects on proliferation, differentiation, survival, and stemness. A subset of PDAC with inactivating mutations in ring finger protein 43 (RNF43) show growth dependency on autocrine WNT ligand signaling and are susceptible to agents that block WNT ligand acylation by Porcupine O-acyltransferase, which is required for proper WNT ligand processing and secretion. For this study, global transcriptomic, proteomic, and metabolomic analyses were performed to explore the therapeutic response of RNF43-mutant PDAC to the Porcupine inhibitor (PORCNi) LGK974. LGK974 disrupted cellular bioenergetics and mitochondrial function through actions that included rapid mitochondrial depolarization, reduced mitochondrial content, and inhibition of oxidative phosphorylation and tricarboxylic acid cycle. LGK974 also broadly altered transcriptional activity, downregulating genes involved in cell cycle, nucleotide metabolism, and ribosomal biogenesis and upregulating genes involved in epithelial-mesenchymal transition, hypoxia, endocytosis, and lysosomes. Autophagy and lysosomal activity were augmented in response to LGK974, which synergistically inhibited tumor cell viability in combination with chloroquine. Autocrine WNT ligand signaling dictates metabolic dependencies in RNF43-mutant PDAC through a combination of transcription dependent and independent effects linked to mitochondrial health and function. Metabolic adaptations to mitochondrial damage and bioenergetic stress represent potential targetable liabilities in combination with PORCNi for the treatment of WNT ligand-addicted PDAC