Engineering an Alkane-Hydroxylating Bacterial Monooxygenase: A Tale of Two Chemistries

Toluene / o-xylene monooxyenase (ToMO) from Pseudomonas sp. OX1 is a multimeric metalloenzyme enzyme that efficiently catalyzes the hydroxylation of aromatic hydrocarbons with high specificity. Though included in a larger group of conserved bacterial multicomponent monooxygenases (BMMs) studied as potential biocatalysts for industrial hydrocarbon chemistry, the substrate specificity and oxygenated intermediates of ToMO differ greatly from its well-characterized, alkane-hydroxylating analog sMMO. Despite a shared global topology and near identical active sites, sMMO can cleave inert C-H bonds in alkanes while ToMO cannot - two seemingly similar structures give rise to vastly different chemistries. This work seeks to determine a structural basis for this difference by mutational analysis of residues thought to conformationally constrain the active site in ToMO, with the goal of replicating the terminal alkane hydroxylating activity of sMMO. To this end, a library of potential alkane-hydroxylating mutants was generated and kinetically characterized, revealing a range of novel behaviors including significant reaction rate enhancements. In combination with low-level computational modeling to quantify the bulk and local rigidity of both sMMOH and ToMOH, we propose a broader strategy for BMM scaffolds to achieve a variety of specific and efficient hydrocarbon chemistries

Formulation and In-vitro Evaluation of Tolterodine Tartrate Tablets by Using High Performance Liquid Chromatographic (HPLC)

Tolterodine tartrate, is a new, potent and competitive muscarinic receptor antagonist in clinical development for the treatment of urge incontinence and other symptoms of unstable bladder. The purpose of this study is to formulation and invitro evaluation of Tolterodine tartrate by high performance liquid chromatography with ultraviolet detection (HPLC-UV). A simple, rapid, and sensitive high-performance liquid chromatographic method was developed and evaluated for invitro formulation of Tolterodine tartrate Tablets. Tablets were analysed by measuring different parameters: lubricated granules content of Tolterodine tartrate having bulk density, tap densities and angle of r content uniformity, assay and related substances. Separation of Tolterodine tartrate was achieved within a single chromatographic run on 5µm 4.6x250mm with UV detection at 280 nm, under isocratic conditions, using Acetonitrile and A mixture of 65 volumes of buffer solution prepared by mixing 2.2 ml of orthophosphoric acid to 1000 ml with water, adjusted to pH 3.0 with triethylamine in 35:65 ratio with a flow rate of 1.5 ml/min. From the results, it was clear that designed formulations among f7 displayed drug release in the range of 55.66% to 102.067% in 10 min, which showed improved invitro dissolution rate compared to other formulations as well as others parameters were found to be good as compared to other formulations. Similarly, the average content of formulation f7 was found to be 104.58% and Related substances should comply the test. Assays of f7 were found to be 96.04%, the limit is 90% - 110% of the label claim having weight variation range from  82.50 mg-91.50 mg. epose And flim coated Tolterodine tartrate tablets having friability, thickness, hardness, weight variation, invitro dissolution

An antibody that prevents serpin polymerisation acts by inducing a novel allosteric behavior

Serpins are important regulators of proteolytic pathways with an antiprotease activity that involves a conformational transition from a metastable to a hyperstable state. Certain mutations permit the transition to occur in the absence of a protease; when associated with an intermolecular interaction, this yields linear polymers of hyperstable serpin molecules, which accumulate at the site of synthesis. This is the basis of many pathologies termed the serpinopathies. We have previously identified a monoclonal antibody (mAb4B12) that, in single-chain form, blocks α1-Antitrypsin (α1-AT) polymerisation in cells. Here, we describe the structural basis for this activity. The mAb4B12 epitope was found to encompass residues Glu32, Glu39 and His43 on helix A and Leu306 on helix I. This is not a region typically associated with the serpin mechanism of conformational change, and correspondingly the epitope was present in all tested structural forms of the protein. Antibody binding rendered β-sheet A - on the opposite face of the molecule - more liable to adopt an 'open' state, mediated by changes distal to the breach region and proximal to helix F. The allosteric propagation of induced changes through the molecule was evidenced by an increased rate of peptide incorporation and destabilisation of a preformed serpin-enzyme complex following mAb4B12 binding. These data suggest that prematurely shifting the β-sheet A equilibrium towards the 'open' state out of sequence with other changes suppresses polymer formation. This work identifies a region potentially exploitable for a rational design of ligands that is able to dynamically influence α1-AT polymerisation

A systematic CRISPR screen defines mutational mechanisms underpinning signatures caused by replication errors and endogenous DNA damage.

Mutational signatures are imprints of pathophysiological processes arising through tumorigenesis. We generated isogenic CRISPR-Cas9 knockouts (Δ) of 43 genes in human induced pluripotent stem cells, cultured them in the absence of added DNA damage, and performed whole-genome sequencing of 173 subclones. ΔOGG1, ΔUNG, ΔEXO1, ΔRNF168, ΔMLH1, ΔMSH2, ΔMSH6, ΔPMS1, and ΔPMS2 produced marked mutational signatures indicative of being critical mitigators of endogenous DNA modifications. Detailed analyses revealed mutational mechanistic insights, including how 8-oxo-dG elimination is sequence-context-specific while uracil clearance is sequence-context-independent. Mismatch repair (MMR) deficiency signatures are engendered by oxidative damage (C>A transversions), differential misincorporation by replicative polymerases (T>C and C>T transitions), and we propose a 'reverse template slippage' model for T>A transversions. ΔMLH1, ΔMSH6, and ΔMSH2 signatures were similar to each other but distinct from ΔPMS2. Finally, we developed a classifier, MMRDetect, where application to 7,695 WGS cancers showed enhanced detection of MMR-deficient tumors, with implications for responsiveness to immunotherapies

Low-input library preparation methods for single molecule sequencing

Over the past two decades, high-throughput DNA sequencing has revolutionized our understanding of epigenetics. The development of quantitative assays that use sequencing to measure chromatin accessibility and methylation state have provided key insights into oncogenesis and cancer metastasis. Recently developed 3rd generation technologies offer significant improvements over current sequencing platforms, including kilobase-scale read lengths and native detection of epigenetic marks on single molecules. Profiling techniques built on these platforms can therefore capture the epigenetic states of single chromatin fibers with unprecedented resolution. However, the inherently higher input requirements for these assays and sequencing platforms have limited their general applicability in medical settings, where sample material is constrained. In this dissertation, we address this problem by developing a new generalizable strategy for preparing native 3rd generation sequencing libraries from low-input samples using a hyperactive transposase. In Chapter 1, we motivate and contextualize this work by discussing how the epigenome is dysregulated in cancer progression and the current tools we use to study it. Then, in Chapter 2 we discuss how our transposase-mediated method enables the study of chromatin fibers in a range of clinically relevant samples including cancer cell lines and patient derived xenograft models. In Chapter 3, we present further methodological improvements that enable direct library preparation from cells and nuclei, collectively lowering input requirements ~20X and making native single molecule profiling studies competitive with existing epigenomic assays. Finally, we conclude in Chapter 4 by considering how our method is a general tool for using 3rd generation sequencing as a read out for biological assays. We demonstrate this for two specific cases: high-depth profiling of targeted genomic regions and resolving chromatin states in single cells

Pelatihan Pengamatan Kupu-Kupu Menggunakan Aplikasi Odolepi Berbasis Android di SMA Muhammadiyah Pamijahan Bogor

This community partnership program is aimed at downstream activities and mentoring training programs for observing the diversity of butterflies and dragonflies at SMA Muhammadiyah Pamijahan, Bogor, West Java. In addition, based on observations from previous research, information about the diversity of dragonflies and butterflies in the Cibodas Botanical Gardens was also produced, a guidebook for observing dragonflies and butterflies in printed and mobile form, named Odolepi. This application can be displayed on android-based mobile phones. Meanwhile, the target in this Service at SMA Muhammadiyah Pamijahan Bogor is an innovation activity that is given in the form of insect observation activities according to the subject matter in biology subjects, namely butterflies by using observation guidelines in the form of mobile or android which can be stored in mobile phones. so it can be used anytime and anywhere. For students who do not have an android mobile phone, the service team will provide a manual in printed form. As for the results of display conformity getting 77.54%, language suitability of 81.87 5 and media suitability of 76.27% it can be concluded that the media used it's been very good. This activity is expected to increase knowledge, skills and of course attitudes to better protect and preserve butterflies which in turn preserves the environment