Theory of Site-Specific DNA-Protein Interactions in the Presence of Conformational Fluctuations of DNA Binding Domains

We develop a theory that explains how the thermally driven conformational fluctuations in the DNA binding domains (DBDs) of the DNA binding proteins (DBPs) are effectively coupled to the one-dimensional searching dynamics of DBPs for their cognate sites on DNA. We show that the rate γ_opt, associated with the flipping of conformational states of DBDs beyond which the maximum search efficiency of DBPs is achieved, varies with the one-dimensional sliding length L as γ_opt ∝ L⁻² and with the number of roadblock protein molecules present on the same DNA m as γ_opt ∝ m². The required free energy barrier E_RTO associated with this flipping transition seems to be varying with L as E_RTO ∝ ln L². When the barrier height associated with the conformational flipping of DBDs is comparable with that of the thermal free energy, then the possible value of L under in vivo conditions seems to be L ≤ 70 bps.     

SMC complexes: Lifting the lid on loop extrusion

Loop extrusion has emerged as a prominent hypothesis for how SMC complexes shape chromosomes – single molecule in vitro observations have yielded fascinating images of this process. When not extruding loops, SMC complexes are known to topologically entrap one or more DNAs. Here, we review how structural insight into the SMC complex cohesin has led to a molecular framework for both activities: a Brownian ratchet motion, associated with topological DNA entry, might repeat itself to elicit loop extrusion. After contrasting alternative loop extrusion models, we explore whether topological loading or loop extrusion is more adept at explaining in vivo SMC complex function. SMC variants that experimentally separate topological loading from loop extrusion will in the future probe their respective contributions to chromosome biology.     

The evolution of porcine embryo in vitro production

The in vitro production of porcine embryos has presented numerous challenges to researchers over the past four decades. Some of the problems encountered were specific to porcine gametes and embryos and needed the concerted efforts of many to overcome. Gradually, porcine embryo in vitro production systems became more reliable and acceptable rates of blastocyst formation were achieved. Despite the significant improvements, the problem of polyspermic fertilization has still not been adequately resolved and the embryo in vitro culture conditions are still considered to be suboptimal. Whereas early studies focused on increasing our understanding of the reproductive processes involved, the technology evolved to the point where in vitro-matured oocytes and in vitro-produced embryos could be used as research material for developing associated reproductive technologies, such as SCNT and embryo cryopreservation. Today, the in vitro procedures used to mature oocytes and culture embryos are integral to the production of transgenic pigs by SCNT. This review discusses the major achievements, advances, and knowledge gained from porcine embryo in vitro production studies and highlights the future research perspectives of this important technology.     

Exposure of Agaricus bisporus to Trichoderma aggressivum f. europaeum leads to growth inhibition and induction of an oxidative stress response

Green mould disease of mushroom, Agaricus bisporus,is caused by Trichodermaspecies and can result in substantial crop losses.Label free proteomic analysis of changes in the abundance of A. bisporusproteins following exposure to T. aggressivumsupernatantin vitroindicated increased abundance of proteins associated with an oxidative stress response (zinc ion binding (+6.6 fold); peroxidase activity (5.3-fold); carboxylic ester hydrolase (+2.4 fold); dipeptidase (+3.2 fold); [2Fe-2S] cluster assembly (+3.3 fold)). Proteins that decreased in relative abundance were associated with growth: structural constituent of ribosome, translation (-12 fold), deadenylation-dependent decapping of nuclear-transcribed mRNA (-3.4 fold), and small GTPase mediated signal transduction (-2.6 fold). In vivoanalysis revealed that 10^-4 T. aggressivuminoculum decreased the mushroom yield by 29% to 56% and 10^-3 T. aggressivuminoculum decreased the mushroom yield by 68% to 100%. Proteins that increased in abundance in A. bisporusin vivofollowing exposure to T. aggressivumindicated an oxidative stress response and included proteins with pyruvate kinase activity (+2.6 fold) and hydrolase activity (+2.1 fold)). The results indicate that exposure of A. bisporusmycelium to T. aggressivum in vitroand in vivoresulted in an oxidative stress response and reduction in growth.     

Control of yeast-mycelium dimorphism in vitro in Dutch elm disease fungi by manipulation of specific external stimuli

Dutch elm disease (DED) fungi exhibit yeast-mycelium dimorphism both in planta and in vitro. However, previously published data on the transition between these two growth forms in vitro were mostly obtained from a single strain. We examined the effect of six factors on yeast-mycelium dimorphism in vitro in ten strains of Ophiostoma ulmi, Ophiostoma novo-ulmi and Ophiostoma himal-ulmi. Nitrogen sources, calcium, and yeast extract, altogether with inhibitors of phosphodiesterase (caffeine) and dioxygenases (propyl gallate and salicylic acid) were tested in defined culture media. Morphological response to manipulation of several of these factors varied according to the strain of Ophiostoma being analysed. Responses ranged from no statistical differences in morphological transitions to stimulation or reversion of yeast-mycelium dimorphism with the treatments that were tested. These results suggest that different mechanisms and pathways operate in the control of the yeast-mycelium transition in DED pathogens. Oxylipins could be involved in the yeast-to-mycelium transition, since the addition of a dioxygenase inhibitor, salicylic acid, reduced mycelium production in all strains that were tested.     

Systems for in vivo hypermutation: a quest for scale and depth in directed evolution

Traditional approaches to the directed evolution of genes of interest (GOIs) place constraints on the scale of experimentation and depth of evolutionary search reasonably achieved. Engineered genetic systems that dramatically elevate the mutation of target GOIs in vivo relieve these constraints by enabling continuous evolution, affording new strategies in the exploration of sequence space and fitness landscapes for GOIs. We describe various in vivo hypermutation systems for continuous evolution, discuss how different architectures for in vivo hypermutation facilitate evolutionary search scale and depth in their application to problems in protein evolution and engineering, and outline future opportunities for the field.     

Antineoplastic activity of the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine in anaplastic large cell lymphoma

DNA methylation is an epigenetic mechanism establishing long-term gene silencing during development and cell commitment, which is maintained in subsequent cell generations. Aberrant DNA methylation is found at gene promoters in most cancers and can lead to silencing of tumor suppressor genes. The DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (5-aza-CdR) is able to reactivate genes silenced by DNA methylation and has been shown to be a very potent epigenetic drug in several hematological malignancies. In this report, we demonstrate that 5-aza-CdR exhibits high antineoplastic activity against anaplastic large cell lymphoma (ALCL), a rare CD30 positive non-Hodgkin lymphoma of T-cell origin. Low dose treatment of ALCL cell lines and xenografted tumors causes apoptosis and cell cycle arrest in vitro and in vivo. This is also reflected in genome-wide expression analyses, where genes related to apoptosis and cell death are amongst the most affected targets of 5-aza-CdR. Furthermore, we observed demethylation and re-expression of p16^INK4A after drug administration and senescence associated β-galactosidase activity. Thus, our data provide evidence that 5-aza-CdR is highly efficient against ALCL and warrants further clinical evaluation for future therapeutic use.     

Integrating cellular dimensions with cell differentiation during early development

Early embryo development is characterized by alteration of cellular dimensions and fating of blastomeres. An emerging concept is that cell size and shape drive cellular differentiation during early embryogenesis in a variety of model organisms. In this review, we summarize recent advances that elucidate the contribution of the physical dimensions of a cell to major embryonic transitions and cell fate specification in vivo. We also highlight techniques and newly evolving methods for manipulating the sizes and shapes of cells and whole embryos in situ and ex vivo. Finally, we provide an outlook for addressing fundamental questions in the field and more broadly uncovering how changes to cell size control decision making in a variety of biological contexts.     

DsRed-mediated oligomerization stabilizes HMGB1 on chromatin in vivo and on DNA in vitro

High-mobility group box-1 (HMGB1) is remarkably mobile in living cells, which reflects its ability to interact only transiently with both DNA and protein. This property is likely essential for HMGB1 nuclear activities. Nonetheless the weak interaction of HMGB1 with DNA and/or protein partners has also been a major limitation for investigating HMGB1 subnuclear localisation and for the identification of HMGB1 containing complexes by conventional biochemical approaches. In the present study, FRAP experiments demonstrated that DsRed-mediated oligomerization strongly reduces HMGB1 mobility due to an increased affinity for cellular chromatin. Moreover, oligomerized DsRed–HMGB1 exhibited a higher affinity for supercoiled DNA in vitro compared to its monomeric counterpart. These results indicate that DsRed-meditated oligomerization is prone to stabilize labile interactions involving HMGB1 both in vivo and in vitro.     

Artificial intelligence for compound pharmacokinetics prediction

Optimisation of compound pharmacokinetics (PK) is an integral part of drug discovery and development. Animal in vivo PK data as well as human and animal in vitro systems are routinely utilised to evaluate PK in humans. In recent years machine learning and artificial intelligence (AI) emerged as a major tool for modelling of in vivo animal and human PK, enabling prediction from chemical structure early in drug discovery, and therefore offering opportunities to guide the design and prioritisation of molecules based on relevant in vivo properties and, ultimately, predicting human PK at the point of design. This review presents recent advances in machine learning and AI models for in vivo animal and human PK for small-molecule compounds as well as some examples for antibody therapeutics.     

Protein phosphorylation regulates in vitro spinach chloroplast petD mRNA 3′-untranslated region stability,processing, and degradation

RNA-binding proteins (RNPs) participate in diverse processes of mRNA metabolism, and phosphorylation changes their binding properties. In spinach chloroplasts, 24RNP and 28RNP are associated with polynucleotide posphorylase forming a complex on charge of pre-mRNA 3′-end maturation. Here, we tested the hypothesis that the phosphorylation status of 24RNP and 28RNP, present in a spinach chloroplast mRNA 3′-UTR processing extract (CPE), controls the transition between petD precursor stabilization, 3′-UTR processing, and RNA degradation in vitro. The CPE processed or stabilized petD precursor depending on the ATP concentration present in an in vitro 3′-UTR processing (IVP) assay. These effects were also observed when ATP was pre-incubated and removed before the IVP assay. Moreover, a dephosphorylated (DP)-CPE degraded petD precursor and recovered 3′-UTR processing or stabilization activities in an ATP concentration dependent manner. To determine the role 24/28RNP plays in regulating these processes a 24/28RNP-depleted (Δ24/28)CPE was generated. The Δ24/28CPE degraded the petD precursor, but when it was reconstituted with recombinant non-phosphorylated (NP)-24RNP or NP-28RNP, the precursor was stabilized, whereas when Δ24/28CPE was reconstituted with phosphorylated (P)-24RNP or P-28RNP, it recovered 3′-UTR processing, indicating that 24RNP or 28RNP is needed to stabilize the precursor, have a redundant role, and their phosphorylation status regulates the transition between precursor stabilization and 3′-UTR processing. A DP-Δ24/28CPE reconstituted or not with NP-24/28RNP degraded petD precursor. Pre-incubation of DP-Δ24/28CPE with NP-24/28RNP plus 0.03 mM ATP recovered 3′-UTR processing activity, and its reconstitution with P-24/28RNP stabilized the precursor. However, pre-incubation of DP-Δ24/28CPE with 0.03 mM ATP, and further reconstitution with NP-24/28RNP or P-24/28RNP produced precursor stability instead of RNA degradation, and RNA processing instead of precursor stability, respectively. Moreover, in vitro phosphorylation of CPE showed that 24RNP, 28RNP, and other proteins may be phosphorylated. Altogether, these results reveal that phosphorylation of 24RNP, 28RNP, and other unidentified CPE proteins mediates the in vitro interplay between petD precursor stability, 3′-UTR processing, and degradation, and support the idea that protein phosphorylation plays an important role in regulating mRNA metabolism in chloroplast.     

Evaluation of an in vitro assay system as a potential alternative to current histamine sensitization test for acellular pertussis vaccines

The histamine sensitization test (HIST) is a lethal test for batch release of acellular pertussis or its combination vaccines (ACV). Large numbers of animals have been used and it is difficult to standardize. Therefore there is an urgent need to develop an in vitro alternative to HIST.An in vitro test system has been developed as a potential alternative to HIST, to examine both the functional domains of PT based on a combination of enzyme coupled-HPLC (E-HPLC) and carbohydrate binding assays. We describe here an international collaborative study, which involved sixteen laboratories from 9 countries to assess the methodology transferability of the in vitro test system and its suitability for the testing of three different types of ACV products that are currently used worldwide. This study also evaluated further the relationship between the in vivo activity by HIST and the in vitro assay system.The results showed that the methodology of the E-HPLC and carbohydrate binding assays are transferable between laboratories worldwide and is suitable for the three types of ACV products included in the study. Although direct correlation between the in vitro assay system and the in vivo HIST (temperature reduction assay) for each individual vaccine lot cannot be established due to the large variation in the HIST results, the observation that the mean estimates of the in vitro and in vivo activities gave the same rank order of the three vaccine types included in the study is encouraging. The in vitro systems provide reproducible product specific profiles which supports their use as a potential alternative to the HIST.     

Discovery of novel pathways for carbohydrate metabolism

Closing the gap between the increasing availability of complete genome sequences and the discovery of novel enzymes in novel metabolic pathways is a significant challenge. Here, we review recent examples of assignment of in vitro enzymatic activities and in vivo metabolic functions to uncharacterized proteins, with a focus on enzymes and metabolic pathways involved in the catabolism and biosynthesis of monosaccharides and polysaccharides. The most effective approaches are based on analyses of sequence-function space in protein families that provide clues for the predictions of the functions of the uncharacterized enzymes. As summarized in this Opinion, this approach allows the discovery of the catabolism of new molecules, new pathways for common molecules, and new enzymatic chemistries.     

Fusarium mycotoxins: Effects on reproductive function in domestic animals—A review

On a global scale, cereal grains and animal feed may be contaminated with trichothecenes, such as deoxynivalenol and T-2 toxin, zearalenone (ZEA), and fumonisins, the major mycotoxins of Fusarium fungi. Of these mycotoxins, ZEA is unequivocally implicated in reproductive disorders of swine and other domestic animals. Experiments in vivo and in vitro indicate that ZEA and its metabolites exert estrogenic effects resulting in functional and morphological alterations in reproductive organs. Recently, the potential of trichothecenes and fumonisins to cause reproductive disorders in domestic animals has been investigated. The present review summarizes the toxicological data on the effects of Fusarium mycotoxins on ovarian function, testicular function, placenta and fetus, and puberty/sexual maturity of domestic animals. The results of in vivo animal studies and in vitro tests are reported and discussed.     

Recent progress in metal-based molecular probes for optical bioimaging and biosensing

Biological imaging and biosensing from subcellular/cellular level to whole body have enabled non-invasive visualisation of molecular events during various biological and pathological processes, giving great contributions to the rapid and impressive advances in chemical biology, drug discovery, disease diagnosis and prognosis. Optical imaging features a series of merits, including convenience, high resolution, good sensitivity, low cost and the absence of ionizing radiation. Among different luminescent probes, metal-based molecules offer unique promise in optical bioimaging and biosensing in vitro and in vivo, arising from their small sizes, strong luminescence, large Stokes shifts, long lifetimes, high photostability and tunable toxicity. In this review, we aim to highlight the design of metal-based molecular probes from the standpoint of synthetic chemistry in the last 2 years for optical imaging, covering d-block transition metal and lanthanide complexes and multimodal imaging agents.     

Bovine in vitro fertilization: In vitro oocyte maturation and sperm capacitation with heparin

As a result of research in the 1980s on in vitro maturation, sperm capacitation, and in vitro fertilization, the bovine is now one of the important models for development. Further, the current production of bovine embryos in vitro rivals that of in vivo embryo production for commercial applications. Researchers of today may be unaware of why decisions were made in the procedures. This review addresses the state of the art at the time of the work by Parrish et al. (Bovine in vitro fertilization with frozen thawed semen. Theriogenology 1986;25:591–600), and how later work would explain success or failure of competing procedures. Important was the use of frozen semen and heparin capacitation, because this allowed future researchers/practitioners to change sperm numbers and capacitation conditions to adjust for variations among bulls. The large numbers of citation of the original work stand the testament of time in the repeatability and success of the procedures. The work was done within the environment of the N.L. First laboratory and the unique interactions with a large number of talented graduate students, postdoctoral researchers, and technicians.