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Post-translational modifications (PTMs) dynamically regulate the critical stress response and tumor suppressive functions of p53. Among these, acetylation events mediated by multiple acetyltransferases lead to differential target gene activation and subsequent cell fate. However, our understanding of these events is incomplete due to, in part, the inability to selectively and dynamically control p53 acetylation. We recently developed a heterobifunctional small molecule system, AceTAG, to direct the acetyltransferase p300/CBP for targeted protein acetylation in cells. Here, we expand AceTAG to leverage the acetyltransferase PCAF/GCN5 and apply these tools to investigate the functional consequences of targeted p53 acetylation in human cancer cells. We demonstrate that the recruitment of p300/CBP or PCAF/GCN5 to p53 results in distinct acetylation events and differentiated transcriptional activities. Further, we show that chemically induced acetylation of multiple hotspot p53 mutants results in increased stabilization and enhancement of transcriptional activity. Collectively, these studies demonstrate the utility of AceTAG for functional investigations of protein acetylation.

Electrochemistry has recently emerged as a powerful approach in small-molecule synthesis owing to its numerous attractive features, including precise control over the fundamental reaction parameters, mild reaction conditions and innate scalability. Even though these advantages also make it an attractive strategy for chemoselective modification of complex biomolecules such as proteins, such applications remain poorly developed. Here we report an electrochemically promoted coupling reaction between 5-hydroxytryptophan (5HTP) and simple aromatic amines—electrochemical labelling of hydroxyindoles with chemoselectivity (eCLIC)—that enables site-specific labelling of full-length proteins under mild conditions. Using genetic code expansion technology, the 5HTP residue can be incorporated into predefined sites of a recombinant protein expressed in either prokaryotic or eukaryotic hosts for subsequent eCLIC labelling. We used the eCLIC reaction to site-specifically label various recombinant proteins, including a full-length human antibody. Furthermore, we show that eCLIC is compatible with strain-promoted alkyne-azide and alkene-tetrazine click reactions, enabling site-specific modification of proteins at two different sites with distinct labels.

ABSTRACTUpregulation of utrophin, the autosomal homologue of dystrophin, can compensate dystrophin deficiency in Duchenne Muscular Dystrophy (DMD) although the therapeutic success is yet to be achieved. The present study has identified Poly (C) binding protein 2 (PCBP2) as a post-transcriptional suppresser for the expression of utrophin-A, the muscle-specific utrophin isoform. This study confirms nuclear retention of utrophin-A mRNA in C2C12 cells, which is mediated by PCBP2. Further investigation demonstrates PCBP2-dependent nuclear retention of follistatin mRNA as well. Its involvement in nuclear retention of mRNA sheds light on a novel function of PCBP2 that makes utrophin-A mRNA less available in cytosol. PCBP2, therefore, may be a target to de-repress utrophin-A expression in DMD.

Site-specific noncanonical amino acid (ncAA) mutagenesis in living cells has traditionally relied on heterologous, nonsense-suppressing aminoacyl-tRNA synthetase (aaRS)/tRNA pairs that do not cross-react with their endogenous counterparts. Such heterologous pairs often perform suboptimally in a foreign host cell since they were not evolutionarily optimized to function in the foreign environment. This suboptimal performance restricts the number of ncAAs that can be simultaneously incorporated into a protein. Here, we show that the use of an endogenous aaRS/tRNA pair to drive ncAA incorporation can offer a potential solution to this limitation. To this end, we developed an engineered Escherichia colistrain (ATMY-C321), wherein the endogenous tyrosyl-tRNA synthetase (TyrRS)/tRNA pair has been functionally replaced with an archaeal counterpart, and the release factor 1 has been removed to eliminate competing termination at the UAG nonsense codons. The endogenous TyrRS/tRNACUATyrpair exhibits remarkably efficient nonsense suppression in the resulting cell, relative to established orthogonal ncAA-incorporation systems in E. coli, allowing the incorporation of an ncAA at up to 10 contiguous sites in a reporter protein. Our work highlights the limitations of orthogonal translation systems using heterologous aaRS/tRNA pairs and offers a potential alternative involving the use of endogenous pairs.

Antibody–drug conjugates (ADCs) have emerged as a powerful class of anticancer therapeutics that enable the selective delivery of toxic payloads into target cells. There is increasing appreciation for the importance of synthesizing such ADCs in a defined manner where the payload is attached at specific permissive sites on the antibody with a defined drug to antibody ratio. Additionally, the ability to systematically alter the site of attachment is important to fine-tune the therapeutic properties of the ADC. Engineered cysteine residues have been used to achieve such site-specific programmable attachment of drug molecules onto antibodies. However, engineered cysteine residues on antibodies often get “disulfide-capped” during secretion and require reductive regeneration prior to conjugation. This reductive step also reduces structurally important disulfide bonds in the antibody itself, which must be regenerated through oxidation. This multistep, cumbersome process reduces the efficiency of conjugation and presents logistical challenges. Additionally, certain engineered cysteine sites are resistant to reductive regeneration, limiting their utility and the overall scope of this conjugation strategy. In this work, we utilize a genetically encoded photocaged cysteine residue that can be site-specifically installed into the antibody. This photocaged amino acid can be efficiently decaged using light, revealing a free cysteine residue available for conjugation without disrupting the antibody structure. We show that this ncAA can be incorporated at several positions within full-length recombinant trastuzumab and decaged efficiently. We further used this method to generate a functional ADC site-specifically modified with monomethyl auristatin F (MMAF).

The first catalytic enantioselective direct aldol reaction of 3‐acetylcoumarins to pyrazole‐4,5‐diones is reported and shown to proceed through noncovalent substrate activation by a quinine‐derived bifunctional tertiary amino‐amide as the catalyst. The densely functionalized products, consisting of an oxygen‐containing quaternary stereocenter and bearing two biologically relevant heterocycles, are generally obtained in high yields with moderate to excellent enantioselectivities. Organocatalysis: Enantioselective direct aldol reaction of 3‐acetyl‐2H‐chromenes with electrophilic pyrazole‐4,5‐diones has been demonstrated by noncovalent catalysis. The products, comprising an oxygen‐containing quaternary stereogenic center and two biologically important heterocyclic frameworks, were obtained in high yields with moderate to excellent enantioselectivities using a bifunctional tertiary amino‐amide as catalyst.

Diazirines are important for photoaffinity labeling, and their photoisomerization is relatively well-known. This work shows how hyperpolarized NMR spectroscopy can be used to characterize an unstable diazo-compound formed via photoisomerization of a 15N2-labeled silyl-ether-substituted diazirine. This diazirine is prepared in a nuclear spin singlet state via catalytic transfer of spin order from para-hydrogen. The active hyperpolarization catalyst is characterized to provide insight into the mechanism. The photochemical isomerization of the diazirine into the diazo-analogue allows the NMR invisible nuclear singlet state of the parent compound to be probed. The identity of the diazo-species is confirmed by trapping with N-phenyl maleimide via a cycloaddition reaction to afford bicyclic pyrazolines that also show singlet state character. The presence of singlet states in the diazirine and the diazo-compound is validated by comparison of experimental nutation behavior with theoretical simulation. The magnetic state lifetime of the diazo-compound is determined as 12 ± 1 s in CD3OD solution at room temperature, whereas its chemical lifetime is measured as 100 ± 5 s by related hyperpolarized NMR studies. Indirect evidence for the generation of the photoproduct para-N2is presented.

Nucleophilic reactivity of deconjugated butyrolactams has been demonstrated for enantioselective Michael additions to α,β-unsaturated aldehydes and ketones. These reactions are catalyzed by diphenylprolinol silyl ether and trans-1,2-diaminocyclohexane-derived bifunctional primary aminothiourea, respectively, producing the Michael adducts with moderate diastereoselectivities and good to excellent enantioselectivities (up to 99:1 er). Unlike in the case of structurally related deconjugated butenolides where vinylogous addition is prevalent, an exclusive α-addition is observed for deconjugated butyrolactams.

This article presents frequentist inference of accelerated life test data of series systems with independent log-normal component lifetimes. The means of the component log-lifetimes are assumed to depend on the stress variables through a linear stress translation function that can accommodate the standard stress translation functions in the literature. An expectation–maximization algorithm is developed to obtain the maximum likelihood estimates of model parameters. The maximum likelihood estimates are then further refined by bootstrap, which is also used to infer about the component and system reliability metrics at usage stresses. The developed methodology is illustrated by analyzing a real as well as a simulated dataset. A simulation study is also carried out to judge the effectiveness of the bootstrap. It is found that in this model, application of bootstrap results in significant improvement over the simple maximum likelihood estimates.

The electricity market is being deregulated all over the world. Deregulation has brought in a variety of trading systems such as day-ahead trading and has also introduced high volatility of electricity prices. The large variability of price increases the risk for the market participants and forces the business houses to look for a forecasting accuracy of about ± 3%. This paper provides a method to predict next-day electricity prices using dynamic regression methodology where the price was regressed on selected demand, as well as supply side variables available in the public domain, and the error has been modelled using ARIMA / SARIMA models. The results were found to be very encouraging with MAPE lying in the range of ± 3.5% in most cases. In order to reduce the complexity associated with developing many models, a clustering methodology was used to group the different hours of the day so as to reduce the number of forecasting models to be fitted. Agglomerative hierarchical clustering with single linkage was used and models for representative hours had the required level of accuracy.

We investigate evolution of quantum correlations in ensembles of two-qubit nuclear spin systems via nuclear magnetic resonance techniques. We use discord as a measure of quantum correlations and the Werner state as an explicit example. We, first, introduce different ways of measuring discord and geometric discord in two-qubit systems and then describe the following experimental studies:(a) We quantitatively measure discord for Werner-like states prepared using an entangling pulse sequence. An initial thermal state with zero discord is gradually and periodically transformed into a mixed state with maximum discord. The experimental and simulated behavior of rise and fall of discord agree fairly well. (b) We examine the efficiency of dynamical decoupling sequences in preserving quantum correlations. In out experimental setup, the dynamical decoupling sequences preserved the traceless parts of the density matrices at high fidelity. But they could not maintain the purity of the quantum states and so were unable to keep the discord from decaying. (c) We observe the evolution of discord for a singlet-triplet mixed state during a radio-frequency. spin-lock. A simple relaxation model describes the evolution of discord, and the accompanying evolution of fidelity of the long-lived singlet state, reasonably well. [ABSTRACT FROM AUTHOR]

We report an experimental demonstration of a quantum delayed-choice experiment via nuclear magnetic resonance techniques. Two spin-1 /2 nuclei from each molecule of a liquid ensemble are used as target and ancilla qubits. The circuit corresponding to the recently proposed quantum delayed-choice setup has been implemented with different states of an ancilla qubit. As expected theoretically, our experiments clearly demonstrate continuous morphing of the target qubit between particle-like and wave-like behaviors. The experimental visibility of the interference patterns shows good agreement with theory. [ABSTRACT FROM AUTHOR]

Stroboscopic spin flips have already been shown to prolong the coherence times of quantum systems under noisy environments. Uhrig's dynamical decoupling scheme provides an optimal sequence for a quantum system interacting with a dephasing bath. Several experimental demonstrations have already verified the efficiency of such dynamical decoupling schemes in preserving single-qubit coherences. In this work we describe the experimental study of Uhrig's dynamical decoupling in preserving two-qubit entangled states using an ensemble of spin-1/2 nuclear pairs in solution state. We find that the performance of odd-order Uhrig sequences in preserving entanglement is superior to both even-order Uhrig sequences and periodic spin-flip sequences. We also find that there exists an optimal order of the Uhrig sequence in which a singlet state can be stored at high correlation for about 30 seconds. [ABSTRACT FROM AUTHOR]

The Escherichia colityrosyl-tRNA synthetase (EcTyrRS)/tRNAEcTyrpair offers an attractive platform for genetically encoding new noncanonical amino acids (ncAA) in eukaryotes. However, challenges associated with a eukaryotic selection system, which is needed to engineer the platform, have impeded its success in the past. Recently, using a facile E. coli-based selection system, we showed that EcTyrRS could be engineered in a strain where the endogenous tyrosyl pair was substituted with an archaeal counterpart. However, significant cross-reactivity between the UAG-suppressing tRNACUAEcTyrand the bacterial glutaminyl-tRNA synthetase limited the scope of this strategy, preventing the selection of moderately active EcTyrRS mutants. Here we report an engineered tRNACUAEcTyrthat overcomes this cross-reactivity. Optimized selection systems based on this tRNA enabled the efficient enrichment of both strongly and weakly active ncAA-selective EcTyrRS mutants. We also developed a wide dynamic range (WiDR) antibiotic selection to further enhance the activities of the weaker first-generation EcTyrRS mutants. We demonstrated the utility of our platform by developing several new EcTyrRS mutants that efficiently incorporated useful ncAAs in mammalian cells, including photoaffinity probes, bioconjugation handles, and a nonhydrolyzable mimic of phosphotyrosine.

Previous publications from our laboratory have shown that methylglyoxal inhibits mitochondrial respiration of malignant and cardiac cells, but it has no effect on mitochondrial respiration of other normal cells [Biswas, Ray, Misra, Dutta and Ray (1997) Biochem. J. 323, 343–348; Ray, Biswas and Ray (1997) Mol. Cell. Biochem. 171, 95–103]. However, this inhibitory effect of methylglyoxal is not significant in cardiac tissue slices. Moreover, post-mitochondrial supernatant (PMS) of cardiac cells could almost completely protect the mitochondrial respiration against the inhibitory effect of methylglyoxal. A systematic search indicated that creatine present in cardiac cells is responsible for this protective effect. Glutathione has also some protective effect. However, creatine phosphate, creatinine, urea, glutathione disulphide and β-mercaptoethanol have no protective effect. The inhibitory and protective effects of methylglyoxal and creatine respectively on cardiac mitochondrial respiration were studied with various concentrations of both methylglyoxal and creatine. Interestingly, neither creatine nor glutathione have any protective effect on the inhibition by methylglyoxal on the mitochondrial respiration of Ehrlich ascites carcinoma cells. The creatine and glutathione contents of several PMS, which were tested for the possible protective effect, were measured. The activities of two important enzymes, namely glyoxalase I and creatine kinase, which act upon glutathione plus methylglyoxal and creatine respectively, were also measured in different PMS. Whether mitochondrial creatine kinase had any role in the protective effect of creatine had also been investigated using 1-fluoro-2,4-dinitrobenzene, an inhibitor of creatine kinase. The differential effect of creatine on mitochondria of cardiac and malignant cells has been discussed with reference to the therapeutic potential of methylglyoxal.

A rapidly growing industrial complex including oil refineries and chemical industries has developed around the coastal area of Haldia port in the district of Midnapore, West Bengal, India. The coastal water is highly polluted with industrial wastes along with petroleum hydrocarbons. The bacteria isolated from the different sites of the coastal waters were Escherichia coli, Alcaligenes, Acinetobacter, Klebsiellaspp., Micrococcusspp., Vibriospp., Pseudomonas aeruginosaand Vibrio parahaemolyticus. The salinity of the water during the time of collection of samples around the port area was 8.2 ppt. Among the isolated organisms, only two isolates, P. aeruginosaand V. parahaemolyticus, showed growth at 300 mM Mg2+ion concentration. However, a 3 mM Mg2+concentration was detected in the coastal water whereas other metal ion concentrations were less than 3×10−5mM. Resistance to Mg2+(300 mM) was determined by a 5.5‐kb plasmid. A large amount of a 40‐kDa outer membrane protein, which was highly soluble in 1 M MgCl2, was isolated from both V. parahaemolyticusand P. aeruginosa. The secretion of proteins in the culture supernatant of V. parahaemolyticuswas highly increased when the cells were grown in the presence of 300 mM Mg2+, whereas very low secretion was observed in the same concentration of Mg2+in the case of P. aeruginosa. Mg2+may act as a specific release factor in protein secretion by V. parahaemolyticusstrains.

The ongoing pandemic due to coronavirus disease, commonly abbreviated as COVID-19, has unleashed a major health crisis across the world. Although multiple vaccines have emerged, large scale vaccination have proven to be a major challenge, especially in developing nations. As a result, early detection still remains a crucial aspect of containing the spread of the virus. The popularly used test for COVID-19 is limited by the availability of test kits and is time-consuming. This has prompted researchers to use chest x-ray (CXR) and chest tomography (CT) scan images of subjects to predict COVID. Many COVID-19 patients also suffer from viral Pneumonia caused by SARS-CoV2 virus. Hence, distinguishing between bacterial and non-COVID Pneumonia is of paramount importance for proper diagnosis of the patients. To this end, in the present work, we have developed a bi-level prediction model of the subjects into normal, Pneumonia and COVID-19 patients by using a shallow learner based classifier on features extracted by VGG19 from the CXR images. The model is used on 3168 images distributed among normal, Pneumonia and COVID classes. We have created a dataset by collating CXR images from various sources like SIRM COVID-19 Database, Chest Imaging (Twitter), COVID-chestxray-dataset and Chest X-Ray Images. The experimental results confirm the superiority of the proposed model (99.26% accuracy) over the best performing single-level classification method (96.74% accuracy). This result is also at par with the many state-of-the-art methods mentioned in literature. The source code is available in the link https://github.com/sdrxc/Bi-level-Prediction-Model-for-Screening-COVID-19-from-Chest-X-ray-Images.

The enantioselective approach to dihydronaphthalenediols (VIIa)‐(VIIb), isoquinolinediol (VIIc), enantiomeric phenolic ether derivative (IX) of natural cis‐6‐deoxy‐4‐hydroxyscytalone, and heterobicyclic conduritol derivative (X) involves the Barrett asymmetric hydroxyallylation method using B‐methoxydiisopinocampheylborane as chiral auxiliary, followed by ring‐closing metathesis (RCM) of the acetonide‐protected diols (V).

A rapidly growing industrial complex including oil refineries and chemical industries has developed around the coastal area of Haldia port in the district of Midnapore, West Bengal, India. The coastal water is highly polluted with industrial wastes along with petroleum hydrocarbons. The bacteria isolated from the different sites of the coastal waters were Escherichia coli, Alcaligenes, Acinetobacter, Klebsiella spp., Micrococcus spp., Vibrio spp., Pseudomonas aeruginosa and Vibrio parahaemolyticus. The salinity of the water during the time of collection of samples around the port area was 8.2 ppt. Among the isolated organisms, only two isolates, P. aeruginosa and V. parahaemolyticus, showed growth at 300 mM Mg2+ ion concentration. However, a 3 mM Mg2+ concentration was detected in the coastal water whereas other metal ion concentrations were less than 3×10−5 mM. Resistance to Mg2+ (300 mM) was determined by a 5.5-kb plasmid. A large amount of a 40-kDa outer membrane protein, which was highly soluble in 1 M MgCl2, was isolated from both V. parahaemolyticus and P. aeruginosa. The secretion of proteins in the culture supernatant of V. parahaemolyticus was highly increased when the cells were grown in the presence of 300 mM Mg2+, whereas very low secretion was observed in the same concentration of Mg2+ in the case of P. aeruginosa. Mg2+ may act as a specific release factor in protein secretion by V. parahaemolyticus strains.