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PCR, Cloning, Restriction Digestion, Ligation, Transformation, Plasmid et al
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Background The diagnostic yield of whole-exome sequencing (WES) varies from 30%–50% among patients with mild to severe neurodevelopmental delay (NDD)/intellectual disability (ID). Routine retrospective reanalysis of undiagnosed patients has increased the total diagnostic yield by 10–15%. Here, we performed proband-only WES of 1065 patients with NDD...
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The current nosological concept of α-synucleinopathies characterized by the presence of Lewy bodies (LBs) includes Parkinson’s disease (PD), Parkinson’s disease dementia (PDD), and dementia with Lewy bodies (DLB), for which the term “Lewy body disease” (LBD) has recently been proposed due to their considerable clinical and pathological overlap. How...
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N ⁶ -methyladenosine (m ⁶ A) epitranscriptional modifications widely exist in RNA, which play critical roles in RNA metabolism and biogenesis processes. Long non-coding RNAs (lncRNAs) are class of non-coding RNAs longer than 200 nucleotides without protein-coding ability. LncRNAs participate in a large number of vital biological progressions. With...
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Background Stickler syndrome (STL) is a rare, clinically and molecularly heterogeneous connective tissue disorder. Pathogenic variants occurring in a variety of genes cause STL, mainly inherited in an autosomal dominant fashion. Autosomal recessive STL is ultra-rare with only four families with biallelic COL9A3 variants reported to date. Results H...
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Clinically relevant animal models are crucial for effective development of therapeutics for peritoneal carcinomatosis (PC). This protocol describes the generation of patient-derived ascites-dependent xenograft (PDADX) models from the cellular component of ascites. The use of routine intraperitoneal injection of the fluid component of ascites is ana...
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Purpose To describe a case of iris metastasis as the initial presentation of clear cell renal cell carcinoma, and to discuss molecular profiling of both the metastasis and primary kidney tumor. Observations We report a patient with blurred vision who underwent ophthalmic examination and was found to have an iris mass, which was excised and diagnos...
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Here, we provide a protocol combing single-cell genomics and functional experiments to study stepwise folliculogenesis. This protocol isolates different developmental stages of mouse ovarian follicles to perform single-cell RNA-seq and in vitro culture, allowing to dissect and validate key molecular events in guiding folliculogenesis, thus bringing...
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TOP1 CAD-seq enables mapping of TOP1 sites of covalent engagement with DNA. The procedure depends upon enrichment of DNA-covalent adducts using chaotropic salts and immunoprecipitation with an antibody specific for TOP1. Here, we describe a step-by-step protocol compatible with Illumina sequencing and bioinformatic pipeline for preliminary data ana...
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The human MUS81-EME1&2 complexes are structure-selective endonucleases that play important roles in DNA damage repair. Here, we describe a protocol to determine the endonuclease activities of MUS81-EME1&2 complexes toward various DNA structures. We co-express MUS81 with EME1 or EME2 and purify the complexes with high purity, and determine their act...
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Here, we provide an optimized RNA-induced silencing complex (RISC) assembly and cleavage protocol in vitro without using radiolabeled RNA. The protocol is useful to characterize the biochemical properties of the RISC. We describe the preparation of RNA probes, the target RNA, and Drosophila cell lysates for RISC assembly assay. We then detail AGO1...
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Membrane contact sites are recognized as critical means of intercompartmental communication. Here, we describe a protocol for engineering and validating a synthetic bridge between the inner and outer mitochondrial membranes to support functioning of the endogenous mitochondrial contact site and cristae organizing system (MICOS). A chimeric protein,...
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Murine trophoblast stem cells (TSCs) have shaped placental research by providing resources for investigating trophoblast subtype specialization. Trophoblast giant cells (TGCs) are large polyploid cells, which undergo repetitive rounds of DNA replication without intervening mitosis by a process called endoreduplication. Endocrine and paracrine funct...
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The analysis of morphological features of neurons derived from human pluripotent stem cells (hPSCs) is important to describe neuronal phenotypes and changes observed throughout development. Using free and easily accessible tools, we describe a protocol for the morphometric quantification of hPSCs-derived neurons in two- and three-dimensions in vitr...
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Obesity is a prevalent metabolic disorder worldwide. Here, we describe a comprehensive protocol using pegylated arginine deiminase (ADI-EPG 20) to apply the concept that arginine depletion induces systemic autophagy to drive whole-body energy metabolism and weight loss in mice. We detail the steps for cohort setup, mouse husbandry, and treatment an...
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Chromatin immunoprecipitation coupled with quantitative PCR (ChIP-qPCR) even with optimization may give low signal-to-background ratio and spatial resolution. Here, we adapted Cleavage Under Targets and Release Using Nuclease (CUT&RUN) (originally developed by the Henikoff group) to develop CUT&RUN-qPCR. By studying the recruitment of selected prot...
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Identification of protein interactors is fundamental to understanding their functions. Here, we describe a modified protocol for tandem affinity purification coupled with mass spectrometry (TAP/MS), which includes two-step purification. We detail the S-, 2×FLAG-, and Streptavidin-Binding Peptide (SBP)- tandem tags (SFB-tag) system for protein purif...
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Multiplexed single-molecule magnetic tweezers (MT) have recently been employed to probe the RNA synthesis dynamics of RNA-dependent RNA polymerases (RdRp). Here, we present a protocol for simultaneously probing the RNA synthesis dynamics of hundreds of single polymerases with MT. We describe the preparation of a dsRNA construct for probing single R...
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Here, we describe protocols to interrogate the binding of the zinc fingers of the transcription factor ZBTB7A to the fetal γ-globin (HBG) promoter. We detail the steps for performing electrophoretic mobility shift assays (EMSAs), X-ray crystallography, and isothermal titration calorimetry (ITC) to explore this interaction. These techniques could re...
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Necroptosis occurs predominantly in the center of late-stage tumors and necroptotic cells are dispersed and difficult to be detected by Western blotting of key markers without enrichment by microdissection. To overcome these obstacles, this protocol provides a detailed immunohistochemistry-oriented approach including the steps of tumor isolation fr...
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Sensory regulation, the ability to select and process sensory information to plan and perform appropriate behaviours, provides a foundation for learning. From early in development, infants manifest differences in the strategies used for sensory regulation. Here, we discuss the nature and characteristics of sensory seeking, a key behavioural strateg...
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Alternative lengthening of telomeres (ALT) is a telomerase-independent but recombination-dependent pathway that maintains telomeres. Here, we describe a protocol to stimulate the formation of ALT-associated PML bodies (APBs) and ALT activity by tethering PML-IV to telomeres in human U2OS cells. Through immunofluorescence, in situ hybridization, and...
Article
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This protocol presents a variation on the 2-ΔΔCt technique for qPCR analysis. Our approach requires the inclusion of a standard curve on each qPCR plate, and like the 2-ΔΔCt technique, is dependent on the stability of housekeeping gene expression. However, unlike the 2-ΔΔCt technique, our approach corrects for imperfect cDNA amplification efficienc...
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During outbreaks, the lack of diagnostic “gold standard” can mask the true burden of infection in the population and hamper the allocation of resources required for control. Here, we present an analytical framework to evaluate and optimize the use of diagnostics when multiple yet imperfect diagnostic tests are available. We apply it to laboratory r...
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Eliminating the risk of bugs and external decryption in cryptographic keys has always been a challenge for researchers. The current research is based on a new design that uses an Omega network-based pseudorandom DNA key generation method to produce cryptographic keys for symmetric key systems. The designed algorithm initially takes two random binar...
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The application of molecular genetic markers in the work-up of various diseases carries a great significance for current clinical and diagnostic practice. This routine only became possible after the finalization of the Human Genome Project with mapping of genes, essential for human pathology. Incorporating high-resolution genetic techniques and a w...
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Desde 2020, a Semana da Biologia UFABC é um evento online, gratuito e com emissão de certificado para os participantes que alcançarem, pelo menos, 75% de presença no evento. A X Semana da Biologia UFABC, realizada de 12 a 23 de Julho de 2021, teve cerca de 3900 inscrições, sendo 34% do estado de São Paulo e 65% graduando de outra instituição. Cerca...
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presentato al 53° Congresso Società Italiana di Storia della Medicina, Senigallia 28-30 aprile 2022, pp. 108-110 Il significato tradizionale di immunità, sia nell'immaginario popolare che in quello medico, è di tipo protettivo-privativo. Si è esenti da qualcosa, da una malattia, dal pagare le tasse, come era previsto per i medici che esercitavano n...
Preprint
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By linking conceptual theories with observed data, generative models can support reasoning in complex situations. They have come to play a central role both within and beyond statistics, providing the basis for power analysis in molecular biology, theory building in particle physics, and resource allocation in epidemiology, for example. We introduc...
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In 2022 new subvariants of Coronavirus appeared in South Africa, and spread rapidly to other parts of the world. The effects of selective evolution on increasing the already high contagiousness of CoV19 up to Omicron were previously discussed with high accuracy using concepts new to molecular biology (from mathematics and physics) that have been de...
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. This essay attempts to reconstruct the story of ptomaines, discovered in 1872 by the Italian chemist Francesco Selmi. This work provides a brief summary of this story, focusing also on an emblematic clinical study by the end of the 19th century, written by Raffaele Canger, a doctor who worked in the Asylum of Nocera Inferiore in Italy. The main t...
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Whilst data on biochemical networks has increased several-fold, our comprehension of the underlying molecular biology is incomplete and inadequate. Simulation studies permit data collation from disparate time points and the imputed trajectories can provide valuable insights into the molecular biology of complex biochemical systems. Although, stocha...
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Moloney murine leukemia virus reverse transcriptase (MMLV-RT) is the most frequently used enzyme in molecular biology for cDNA synthesis. To date, reverse transcription coupled with Polymerase Chain Reaction, known as RT-PCR, has been popular as an excellent approach for the detection of SARS-CoV-2 during the COVID-19 pandemic. In this study, we ai...
Preprint
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Background Kernel size-related traits, as critical determinants of wheat kernel weight and yield potential, are complex quantitative traits controlled by polygenes. Genome-wide identification of important and stable quantitative trait loci (QTL) and functional genes for these traits can advance molecular genetic improvement of wheat kernel yield....
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The unique geography of Chile encompasses a wide diversity of ecosystems and a rich biodiversity. However, the platyhelminth fauna has been poorly studied. The aim of this work is to compile the historical record of freshwater planarians described for this country. We accessed worldwide databases and published articles to provide a comprehensive re...
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In hemophilia A (HA), F8 nonsense variants, and particularly those affecting the large factor VIII (FVIII) B domain that is dispensable for coagulant activity, display lower association with replacement therapy-related anti-FVIII inhibitory antibodies as retrieved from multiple international databases. Since null genetic conditions favour inhibitor...
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Background Hand, foot, and mouth disease (HFMD) is a common child infectious disease caused by more than 20 enterovirus (EV) serotypes. In recent years, enterovirus A71 (EV-A71) has been replaced by Coxsackievirus A6 (CV-A6) to become the predominant serotype. Multiple EV serotypes co-circulate in HFMD epidemics, and this study aimed to investigate...
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Tuberculosis (TB) is the leading cause of death from any bacterial infection, causing 1.5 million deaths worldwide each year. Due to the emergence of drug-resistant strains of Mycobacterium tuberculosis (Mtb) there have been significant efforts aimed at developing novel drugs to treat TB. One promising drug target in Mtb is the arabinogalactan bios...
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The Oncaeidae is an important family of small copepods in marine ecosystems and is characterized by high diversity and abundance. The family was established by Giesbrecht in the late 19 th century. Since then, 115 oncaeid species have been discovered and described. However, Oncaeidae species are small-sized and show considerable morphological simil...
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Rhabdomyosarcomas (RMS) are mesenchyme-derived tumors and the most common childhood soft tissue sarcomas. Treatment is intense, with a nevertheless poor prognosis for high-risk patients. Discovery of new therapies would benefit from additional preclinical models. Here, we describe the generation of a collection of 19 pediatric RMS tumor organoid (t...
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Clioquinol and nitroxoline, two drugs with numerous pharmacological properties fallen into disuse for many decades. The first was considered dangerous due to contraindications and the second mainly because was taken as ineffective, despite its known antibacterial activity. In the last decades, the advances in pharmaceutical chemistry, molecular bio...
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Atypical spindle cell/pleomorphic lipomatous tumor (ASCPT) constitutes an emerging entity of lipomatous tumors. It is a benign tumor. It occurs typically in limbs and limb girdles. The occurrence in oral cavity is unusual. The diagnosis of this neoplasm is challenging. Herein, we report a case of ASCPT arising in the tongue. ASCPT is a benign adipo...
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Info-autopoiesis or the self-referenced, recursive, interactive process of information self-production that engages all living beings in their efforts to satisfy their physiological and/or relational needs relies on Bateson's difference which makes a difference. Living beings, as active manipulators/observers of their environment, derive meaning fr...
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Medulloblastoma is currently subclassified into distinct DNA methylation subgroups/subtypes with particular clinico-molecular features. Using RNA sequencing (RNA-seq) in large, well-annotated cohorts of medulloblastoma, we show that transcriptionally group 3 and group 4 medulloblastomas exist as intermediates on a bipolar continuum between archetyp...
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Intrinsically disordered proteins and regions (IDPs and IDRs) and their importance in biology are becoming increasingly recognized in biology, biochemistry, molecular biology and chemistry textbooks, as well as in current protein science and structural biology curricula. We argue that the sequence → dynamic conformational ensemble → function princi...
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Anastomosing haemangioma (AH) is a newly described distinct vascular neoplasm that histologically may confuse with well-differentiated angiosarcoma (AS) for those who are unfamiliar with this rare entity. We aimed to identify molecular genetic differences between AHs and ASs by carrying out immunohistochemistry (IHC), fluorescence in situ hybridiza...
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Fluorescent proteins are essential reporters in cell and molecular biology. Here, we found that red-fluorescent proteins possess an alternative translation initiation site that produces a short functional protein isoform in both prokaryotes and eukaryotes. The short isoform creates significant background fluorescence that biases the outcome of expr...
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The global burden of foodborne disease is enormous and foodborne pathogens are the leading cause of human illnesses. The detection of foodborne pathogenic bacteria has become a research hotspot in recent years. Rapid detection methods based on immunoassay, molecular biology, microfluidic chip, metabolism, biosensor, and mass spectrometry have devel...
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The paper continues a series of publications devoted to the new finds of macrofungi (ascomycetes, basidiomycetes) in regions of the Russian Far East. A total of 61 species and intraspecific taxa of macromycetes are reported for the first time from 6 administrative units of the Russian Far East: Amur Oblast, Jewish Autonomous Oblast, Magadan Oblast,...
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Over the two years of the novel coronavirus infection (COVID-19) pandemic, there has been an evolution of views in various fields of medicine, which has led to a powerful development of scientific research in the field of epidemiology, clinic, diagnosis and therapy of COVID-19. The article highlights the evolution of views and approaches to the stu...
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Introducción. La infección genital por Chlamydia trachomatis es una de las más frecuentes en el mundo, en este sentido, cada año se registran cerca de 85 millones de nuevos casos de esta patología que cursa con severas complicaciones en la mujer y recién nacido. Objetivo. Determinar las características clínico-epidemiológicas de la infección por C....
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Combining ability along with heterosis was elucidated in the 21 F1 hybrid population of rice derived from a 7 × 7 diallel mating design. Furthermore, to formulate a strategy for marker-based prediction of heterosis, the molecular genetic diversity (GD) was then surveyed among the seven parental lines with RAPD markers. Analysis of combining ability...
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Retrotransposons account for more than one-third of the pig reference genome. On account of the genome variability in different breeds, structural variation (SV) caused by retrotrans-poson-generated deletion or insertion may have a function in the genome. Litter size is one of the most important reproductive traits and significantly impacts profita...
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Molecular genetic engineering technologies such as CRISPR/Cas9 have made the accurate and safe genetic engineering of human embryos possible. Further advances in genomics have isolated genes that predict qualities and traits associated with intelligence. Given these advances, prospective parents could use these biotechnologies to genetically engine...
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For nearly a century the main focus in biological disciplines such as molecular biol- ogy, biochemistry, genetics and evolutionary theory was cellular life as a machine like process in which mechanistic pathways regulate metab- olism, genetic reading and translation into pro- teins and evolution by variations (random error replications) and selecti...
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In manually advancing experimental processes, the stages may be long-term and need to be repeated. Human errors with the repetition of the steps turn into a time-consuming and high-cost for the experiment processes. For this reason, autonomous liquid processing systems are promising technologies. However, in addition to the high cost of fully autom...