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P-glycoprotein (P-gp) plays a crucial role in cellular detoxification and drug efflux processes, transitioning between inward-facing (IF) open, occluded, and outward-facing (OF) states to facilitate substrate transport. Its role is critical in cancer therapy, where P-gp contributes to the multidrug resistance phenotype. In our study, classical and enhanced molecular dynamics (MD) simulations were conducted to dissect the structural and functional features of the P-gp conformational states. Our advanced MD simulations, including kinetically excited targeted MD (ketMD) and adiabatic biasing MD (ABMD), provided deeper insights into state transition and translocation mechanisms. Our findings suggest that the unkinking of TM4 and TM10 helices is a prerequisite for correctly achieving the outward conformation. Simulations of the IF-occluded conformations, characterized by kinked TM4 and TM10 helices, consistently demonstrated altered communication between the transmembrane domains (TMDs) and nucleotide binding domain 2 (NBD2), suggesting the implication of this interface in inhibiting P-gp's efflux function. A particular emphasis was placed on the unstructured linker segment connecting the NBD1 to TMD2 and its role in the transporter's dynamics. With the linker present, we specifically noticed a potential entrance of cholesterol (CHOL) through the TM4-TM6 portal, shedding light on crucial residues involved in accommodating CHOL. We therefore suggest that this entry mechanism could be employed for some P-gp substrates or inhibitors. Our results provide critical data for understanding P-gp functioning and developing new P-gp inhibitors for establishing more effective strategies against multidrug resistance., Competing Interests: The authors declare no conflict of interest., (© 2024 The Authors.)

Background: Bacterial infection of embryo culture medium is rare but may be detrimental. The main source of embryo culture contamination is semen. Assisted reproduction centers currently lack consensus regarding the methods for preventing and managing embryo culture infection. In our recent case, a successful pregnancy was achieved with intracytoplasmic sperm injection after failed conventional in vitro fertilization owing to bacterial contamination., Case Presentation: We present a case report of two consecutive in vitro fertilization-intracytoplasmic sperm injection cycles with photo and video documentation of the bacterial growth. A 36-year-old Hungarian woman and her 37-year-old Hungarian partner came to our department. They had two normal births followed by 2 years of infertility. The major causes of infertility were a closed fallopian tube and asthenozoospermia. Bacterial infection of the embryo culture medium was observed during in vitro fertilization and all oocytes degenerated. The source was found to be the semen. To prevent contamination, intracytoplasmic sperm injection was used for fertilization in the subsequent cycle. Intracytoplasmic bacterial proliferation was observed in one of the three fertilized eggs, but two good-quality embryos were successfully obtained. The transfer of one embryo resulted in a successful pregnancy and a healthy newborn was delivered., Conclusion: Intracytoplasmic sperm injection may be offered to couples who fail conventional in vitro fertilization treatment owing to bacteriospermia, as it seems to prevent infection of the embryo culture. Even if bacterial contamination appears, our case encourages us to continue treatment. Nevertheless, the development of new management guidelines for the prevention and management of bacterial contamination is essential., (© 2024. The Author(s).)

Drug metabolism and transport, orchestrated by drug-metabolizing enzymes (DMEs) and drug transporters (DTs), are implicated in drug-drug interactions (DDIs) and adverse drug reactions (ADRs). Reliable and precise predictions of DDIs and ADRs are critical in the early stages of drug development to reduce the rate of drug candidate failure. A variety of experimental and computational technologies have been developed to predict DDIs and ADRs. Recent artificial intelligence (AI) approaches offer new opportunities for better predicting and understanding the complex processes related to drug metabolism and transport. We summarize the role of major DMEs and DTs, and provide an overview of current progress in computational approaches for the prediction of drug metabolism, transport, and DDIs, with an emphasis on AI including machine learning (ML) and deep learning (DL) modeling., Competing Interests: Declaration of interests The authors declare no conflicts of interest., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Sulfotransferases (SULTs) are phase II metabolizing enzymes catalyzing the sulfoconjugation from the co-factor 3'-Phosphoadenosine 5'-Phosphosulfate (PAPS) to a wide variety of endogenous compounds, drugs and natural products. Although SULT1A1 and SULT1A3 share 93% identity, SULT1A1, the most abundant SULT isoform in humans, exhibits a broad substrate range with specificity for small phenolic compounds, while SULT1A3 displays a high affinity toward monoamine neurotransmitters like dopamine. To elucidate the factors determining the substrate specificity of the SULT1 isoenzymes, we studied the dynamic behavior and structural specificities of SULT1A1 and SULT1A3 by using molecular dynamics (MD) simulations and ensemble docking of common and specific substrates of the two isoforms. Our results demonstrated that while SULT1A1 exhibits a relatively rigid structure by showing lower conformational flexibility except for the lip (loop L1), the loop L2 and the cap (L3) of SULT1A3 are extremely flexible. We identified protein residues strongly involved in the recognition of different substrates for the two isoforms. Our analyses indicated that being more specific and highly flexible, the structure of SULT1A3 has particularities in the binding site, which are crucial for its substrate selectivity.

Introduction: Intracytoplasmic sperm injection (ICSI) was introduced to achieve fertilization in cases of severe male factor infertility. However, ICSI is often used in cases of non-male factor infertility, such as advanced maternal age or low oocyte number, but the clinical benefit of the method in these indications has not been proven., Material and Methods: A prospective randomized study was conducted in a university clinic between 2018 and 2020. Patients with ≥40 years of age and/or ≤4 oocytes with non-sever male factor infertility were randomized into conventional IVF or ICSI groups. Fertilization rate, embryo quality, implantation, clinical pregnancy and live birth rates were compared., Results: A total of 336 IVF cycles (169 conventional IVF and 167 ICSI) were involved in the study. The fertilization rate was higher in the conventional IVF group compared to the ICSI group (IVF: 61.7%, ICSI: 53.4%, P=0.001). Embryo development and morphology did not show considerable difference between groups. Implantation, clinical pregnancy and live birth rate were 13.1%, 24.3% and 11.4% in the conventional IVF and 10.4%, 19.0%, 12.0% in the ICSI group. The differences were not significant. Subgroup analysis showed a significantly better clinical outcome following conventional IVF when advanced maternal age was accompanied by low oocyte number (Implantation: 11.7% vs 2.6%, P=0.027; Clinical pregnancy: 18.5% vs 4%, P=0.020)., Discussion: A significantly higher fertilization rate, a tendency for higher clinical pregnancy rate was found in conventional IVF treatments compared to ICSI. When advanced maternal age was associated with low oocyte number, ICSI resulted in a substantially lower chance of fertilization and clinical pregnancy. These data suggest that ICSI offers no advantage over conventional IVF in terms of fertilization, embryo quality, implantation and pregnancy rates for couples with advanced maternal age or with low oocyte number., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Patient symptom relief is often heavily influenced by the residence time of the inhibitor-target complex. For the human muscarinic receptor 3 (hMR3), tiotropium is a long-acting bronchodilator used in conditions such as asthma or chronic obstructive pulmonary disease (COPD). The mechanistic insights into this inhibitor remain unclear; specifically, the elucidation of the main factors determining the unbinding rates could help develop the next generation of antimuscarinic agents. Using our novel unbinding algorithm, we were able to investigate ligand dissociation from hMR3. The unbinding paths of tiotropium and two of its analogues, N -methylscopolamin and homatropine methylbromide, show a consistent qualitative mechanism and allow us to identify the structural bottleneck of the process. Furthermore, our machine learning-based analysis identified key roles of the ECL2/TM5 junction involved in the transition state. Additionally, our results point to relevant changes at the intracellular end of the TM6 helix leading to the ICL3 kinase domain, highlighting the closest residue L482. This residue is located right between two main protein binding sites involved in signal transduction for hMR3's activation and regulation. We also highlight key pharmacophores of tiotropium that play determining roles in the unbinding kinetics and could aid toward drug design and lead optimization.

Objectives: During human in vitro fertilisation (IVF) treatments, embryologists attempt to select the most viable embryos for embryo transfer (ET). Previously, embryos were evaluated based on light microscopic morphological parameters. However, this is currently accomplished by morphokinetic analysis of time-lapse recordings. This technique provides us the opportunity to observe cytoplasmic strings at the blastocyst stage. The aim of this work was to examine the relationship between the presence of cytoplasmic strings (CS) and the embryo viability in human in vitro fertilised embryos. Study design: Herein, we present an evaluation of the morphokinetic data on the development of embryos obtained during IVF treatments performed at the Division of Assisted Reproduction between December 2020 and March 2021. The dynamics of embryo development, embryo morphology, and morphokinetic scores generated by a time-lapse system were compared between the presence of cytoplasmic strings (CS+) and their absence (CS-) at the blastocyst stage. Results: The development of 208 embryos from 78 patients was examined. Moreover, 81.2% of the embryos had CS in the blastocyst stage; 77% of CS existed in embryos created by conventional IVF, while 86% of CS existed in embryos fertilised by intracytoplasmic sperm injection (ICSI) ( p = 0.08). A greater number of CS+ embryos developed into a higher quality blastocyst (52.1% vs. 20.5%, p = 0.02). The morphokinetic score values characterising the development of embryos, such as Known Implantation Data Score (KIDScore) and Intelligent Data Analysis (iDAScore), were higher in CS+ groups (KID: 6.1 ± 2.1 vs. 4.7 ± 2.07; iDA: 8.0 ± 1.9 vs. 6.8 ± 2.3, p < 0.01). The dynamics of the early embryo development were similar between the two groups; however, CS+ embryos reached the blastocyst stage significantly earlier (tB: 103.9 h vs. tB: 107.6 h; p = 0.001). Conclusion: Based on our results, the number of embryos with cytoplasmic strings was higher than that without cytoplasmic strings, and its presence is not related to the fertilisation method. These embryos reached the blastocyst stage earlier, and their morphokinetic (KIDScore and iDAScore) parameters were better. All these results suggest that the presence of CS indicates higher embryo viability. The examination of this feature may help us make decisions about the embryos with higher implantation potential., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Joo, Nemes, Dudas, Berkes-Bara, Murber, Urbancsek and Fancsovits.)

The small GTPase Ran is the main regulator of the nucleo-cytoplasmic import and export through the nuclear pore complex. It functions as a molecular switch cycling between the GDP-bound inactive and GTP-bound active state. It consists of a globular (G) domain and a C-terminal region, which is bound to the G-domain in the inactive, GDP-bound states. Crystal structures of the GTP-bound active form complexed with Ran binding proteins (RanBP) show that the C-terminus undergoes a large conformational change, embracing Ran binding domains (RanBD). Whereas in the crystal structures of macromolecular complexes not containing RanBDs the structure of the C-terminal segment remains unresolved, indicating its large conformational flexibility. This movement could not have been followed either by experimental or simulation methods. Here, starting from the crystal structure of Ran in both GDP- and GTP-bound forms we show how rigid the C-terminal region in the inactive structure is during molecular dynamics (MD) simulations. Furthermore, we show how MD simulations of the active form are incapable of mapping the open conformations of the C-terminus. By using the MDeNM (Molecular Dynamics with excited Normal Modes) method, we were able to widely map the conformational surface of the C-terminus of Ran in the active GTP-bound form, which allows us to envisage how it can embrace RanBDs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Czigleczki, de Resende Lara, Dudas, Jang, Perahia, Nussinov and Balog.)

UDP-glucuronosyltransferases (UGTs) are responsible for 35% of the phase II drug metabolism. In this study, we focused on UGT1A1, which is a key UGT isoform. Strong inhibition of UGT1A1 may trigger adverse drug/herb-drug interactions, or result in disorders of endobiotic metabolism. Most of the current machine learning methods predicting the inhibition of drug metabolizing enzymes neglect protein structure and dynamics, both being essential for the recognition of various substrates and inhibitors. We performed molecular dynamics simulations on a homology model of the human UGT1A1 structure containing both the cofactor- (UDP-glucuronic acid) and substrate-binding domains to explore UGT conformational changes. Then, we created models for the prediction of UGT1A1 inhibitors by integrating information on UGT1A1 structure and dynamics, interactions with diverse ligands, and machine learning. These models can be helpful for further prediction of drug-drug interactions of drug candidates and safety treatments., Competing Interests: The authors declare no competing interests., (© 2022 The Author(s).)

Morphological and pharmacological studies indicate that hypothalamic neuropeptide Y (NPY) and proopiomelanocortin (POMC) neurons communicate with each other in rats and regulate a variety of hypothalamic and extrahypothalamic functions. Indeed, electron microscopic studies revealed NPY-immunoreactive (NPI-IR) synapses on β-endorphin-IR neurons in the hypothalamus. However, no such connections have been reported in humans. Here, we studied the putative NPY-β-endorphin associations with high-resolution light microscopic double-label immunocytochemistry in the human hypothalamus. The majority of β-endorphin-IR perikarya appear to be innervated by abutting NPY-IR fibers in the infundibulum/median eminence, receiving more than 6 contacts (38% of the counted neurons) or three to six contacts (42% of the counted neurons). The rest of the β-endorphin-IR neurons are lightly innervated by NPY fibers (14%, one-three contacts) or do not receive any detectable NPY-IR axon varicosities (6% of the counted neurons). Since β-endorphin is cleaved from the proopiomelanocortin (POMC) precursor, the NPY-β-endorphin connections also provide the foundation for NPY-α-MSH and NPY-ACTH connections and their subsequent physiology. The close anatomical connections between NPY-IR nerve terminals and β-endorphin-IR neurons reported herein may represent functional synapses and provide the foundation for NPY-stimulated β-endorphin release. By interacting with β-endorphin, NPY may have a more widespread regulatory capacity than acting alone on different neurotransmitter systems., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Recent years have seen several hybrid simulation methods for exploring the conformational space of proteins and their complexes or assemblies. These methods often combine fast analytical approaches with computationally expensive full atomic molecular dynamics (MD) simulations with the goal of rapidly sampling large and cooperative conformational changes at full atomic resolution. We present here a systematic comparison of the utility and limits of four such hybrid methods that have been introduced in recent years: MD with excited normal modes (MDeNM), collective modes-driven MD (CoMD), and elastic network model (ENM)-based generation, clustering, and relaxation of conformations (ClustENM) as well as its updated version integrated with MD simulations (ClustENMD). We analyzed the predicted conformational spaces using each of these four hybrid methods, applied to four well-studied proteins, triosephosphate isomerase (TIM), 3-phosphoglycerate kinase (PGK), HIV-1 protease (PR) and HIV-1 reverse transcriptase (RT), which provide extensive ensembles of experimental structures for benchmarking and comparing the methods. We show that a rigorous multi-faceted comparison and multiple metrics are necessary to properly assess the differences between conformational ensembles and provide an optimal protocol for achieving good agreement with experimental data. While all four hybrid methods perform well in general, being especially useful as computationally efficient methods that retain atomic resolution, the systematic analysis of the same systems by these four hybrid methods highlights the strengths and limitations of the methods and provides guidance for parameters and protocols to be adopted in future studies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Kaynak, Krieger, Dudas, Dahmani, Costa, Balog, Scott, Doruker, Perahia and Bahar.)

Introduction: Substance P (SP) is a member of the tachykinin family. In the central nervous system, SP participates among others, in the regulation of pain, learning, memory, emotion, and sexual functions. In the periphery, SP affects the gastrointestinal, cardiovascular, and urinary systems. Galanin, similarly to SP, appears to be involved in wide range of physiologic functions, including cognition, waking and sleep, feeding, mood, blood pressure, reproduction, and development, where acts as a trophic factor. The similar distribution of SP-immunoreactive (SP-IR) fibers and galanin-IR perikarya in the human hypothalamus suggests functional interaction between these neuropeptides. Methods: We have utilized double-label immunohistochemistry to reveal these putative juxtapositions. Results: The majority of galanin-IR neurons receive contacting SP-IR fibers that often cover a significant area of the galaninergic perikarya forming multiple en passant type contacts. These SP-galanin juxtapositions are located mainly in the basal part of the infundibulum/median eminence, populating the basal periventricular region as well as the basal perifornical area. Discussion: The density and the morphology of these associations suggest that these contacts are functional synapses and therefore may represent the morphological substrate of the control of SP on multiple functions regulated/modulated by galanin. SP via galanin may modulate anterior pituitary hormone secretion, as contrary to SP, high density of galanin immunoreactivity is present in the median eminence, and by innervating galanin-IR neurons projecting to other parts of the brain, SP can modulate indirectly their activities. Impact statement The present study is the first describing juxtapositions between the substance P (SP)-immunoreactive (IR) and galanin-IR neurons in the human hypothalamus. These juxtapositions may be functional synapses and they may represent the morphological substrate of the control of SP on the galaninergic system. SP via galanin may modulate anterior pituitary hormone secretion, as contrary to SP, high density of galanin immunoreactivity is present in the median eminence. Galanin, released into the hypothalamo-hypophyseal circulation, can reach the anterior pituitary and function as a hypophysiotropic substance and regulates anterior pituitary hormone secretion. SP by innervating galanin-IR neurons, which project to other parts of the brain, can modulate indirectly their activities.

Sulfotransferases (SULTs) are phase II drug-metabolizing enzymes catalyzing the sulfoconjugation from the co-factor 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to a substrate. It has been previously suggested that a considerable shift of SULT structure caused by PAPS binding could control the capability of SULT to bind large substrates. We employed molecular dynamics (MD) simulations and the recently developed approach of MD with excited normal modes (MDeNM) to elucidate molecular mechanisms guiding the recognition of diverse substrates and inhibitors by SULT1A1. MDeNM allowed exploring an extended conformational space of PAPS-bound SULT1A1, which has not been achieved up to now by using classical MD. The generated ensembles combined with docking of 132 SULT1A1 ligands shed new light on substrate and inhibitor binding mechanisms. Unexpectedly, our simulations and analyses on binding of the substrates estradiol and fulvestrant demonstrated that large conformational changes of the PAPS-bound SULT1A1 could occur independently of the co-factor movements that could be sufficient to accommodate large substrates as fulvestrant. Such structural displacements detected by the MDeNM simulations in the presence of the co-factor suggest that a wider range of drugs could be recognized by PAPS-bound SULT1A1 and highlight the utility of including MDeNM in protein-ligand interactions studies where major rearrangements are expected.