Human Homologue Research Articles

Characterization of Dog Glutathione Transferase P1-1, an Enzyme Relevant to Veterinary Medicine.

Glutathione transferases (GSTs) form a family of detoxication enzymes instrumental in the inactivation and elimination of electrophilic mutagenic and carcinogenic compounds. The Pi class GST P1-1 is present in most tissues and is commonly overexpressed in neoplastic cells. GST P1-1 in the dog, Canis lupus familiaris, has merits as a marker for tumors and as a target for enzyme-activated prodrugs. We produced the canine enzyme CluGST P1-1 by heterologous bacterial expression and verified its cross-reactivity with antihuman-GST P1-1 antibodies. The catalytic activity with alternative substrates of biological significance was determined, and the most active substrate found was benzyl isothiocyanate. Among established GST inhibitors, Cibacron Blue showed positive cooperativity with an IC50 value of 43 nM. Dog GST P1-1 catalyzes activation of the prodrug Telcyta, but the activity is significantly lower than that of the human homolog.

Identification of potential inhibitors of Schistosoma mansoni purine nucleoside phosphorylase from neolignan compounds using molecular modelling approaches

Schistosomiasis is a parasitic disease that is part of the neglected tropical diseases (NTDs), which cause significant levels of morbidity and mortality in millions of people throughout the world. The enzyme purine nucleoside phosphorylase from Schistosoma mansoni (SmPNP) represents a potential target for discovering new agents, and neolignans stand out as an important class of compounds. In this work, molecular modeling studies and biological assays of a set of neolignans were conducted against the PNP enzymes of the parasite and the human homologue (HssPNP). The results of the molecular docking described that the neolignans showed good complementarity by the active site of SmPNP. Molecular dynamics (MD) studies revealed that both complexes (Sm/HssPNP - neolignan compounds) were stable by analyzing the root mean square deviation (RMSD) values, and the binding free energy values suggest that the selected structures can interact and inhibit the catalytic activity of the SmPNP. Finally, the biological assay indicated that the selected neolignans presented a better molecular profile of inhibition compared to the human enzyme, as these ligands did not have the capacity to inhibit enzymatic activity, indicating that these compounds are promising candidates and that they can be used in future research in chemotherapy for schistosomiasis. Communicated by Ramaswamy H. Sarma

NKR-P1B Receptor Expression and Function in the Different Innate Lymphoid Cells of the Lung

Group 2 innate lymphoid cells (ILC2) are the majority of ILCs in murine lungs at steady state. ILC2s are the main producer of type-2-cytokines, IL-4, IL-5, IL-9, IL-13, and amphiregulin, playing key roles in lung tissue homeostasis, airway responses to pathogens and allergens, and in cancer-related defenses. ILC functions are regulated by cell surface receptors. NKR-P1B is an inhibitory receptor, which recognizes C-type lectin-related protein (Clr-b) as its ligand. NKR-P1B is expressed on subsets of natural killer cells, ILC2, ILC3, γδ T cells, macrophages and dendritic cells in a tissue-specific manner and regulates NK cell and ILC3 functions in the gut. Expression and function of NKR-P1B in the lung ILC populations is unexplored. Moreover, Clr-b, the ligand for NKR-P1B, is expressed in the bronchial epithelium, endothelial cells and in lung parenchyma, but its role in immune regulation in the lung is unknown. We hypothesize that ILC2s in the lung express NKR-P1B, and their function is regulated by the NKR-P1B:Clr-b recognition system. Using wild-type (WT) and NKR-P1B-deficient mice, we study the expression of NKR-P1B on lung ILC2, and the function of NKR-P1B:Clr-b recognition system in ILC2 development and function. We compare the phenotype, frequency, numbers and cytokine production by ILC2s upon stimulation between WT and NKR-P1B-deficient mice using antibody staining and flow cytometry analysis. This study will reveal the role of NKR-P1B as a model system for its human homolog, NKR-P1A, in the regulation of ILC development and function, advancing our understanding of how immune responses in the lung are regulated.

CarcSeq Measurement of Rat Mammary Cancer Driver Mutations and Relation to Spontaneous Mammary Neoplasia.

The ability to deduce carcinogenic potential from subchronic, repeat dose rodent studies would constitute a major advance in chemical safety assessment and drug development. This study investigated an error-corrected NGS method (CarcSeq) for quantifying cancer driver mutations (CDMs) and deriving a metric of clonal expansion predictive of future neoplastic potential. CarcSeq was designed to interrogate subsets of amplicons encompassing hotspot CDMs applicable to a variety of cancers. Previously, normal human breast DNA was analyzed by CarcSeq and metrics based on mammary-specific CDMs were correlated with tissue donor age, a surrogate of breast cancer risk. Here we report development of parallel methodologies for rat. The utility of the rat CarcSeq method for predicting neoplastic potential was investigated by analyzing mammary tissue of 16-week-old untreated rats with known differences in spontaneous mammary neoplasia (Fischer 344, Wistar Han, and Sprague Dawley). Hundreds of mutants with mutant fractions ≥ 10-4 were quantified in each strain, most were recurrent mutations, and 42.5% of the nonsynonymous mutations have human homologs. Mutants in the mammary-specific target of the most tumor-sensitive strain (Sprague Dawley) showed the greatest nonsynonymous/synonymous mutation ratio, indicative of positive selection consistent with clonal expansion. For the mammary-specific target (Hras, Pik3ca, and Tp53 amplicons), median absolute deviation correlated with percentages of rats that develop spontaneous mammary neoplasia at 104 weeks (Pearson r = 1.0000, 1-tailed p = .0010). Therefore, this study produced evidence CarcSeq analysis of spontaneously occurring CDMs can be used to derive an early metric of clonal expansion relatable to long-term neoplastic outcome.

SARS-CoV-2 Nsp16 activation mechanism and a cryptic pocket with pan-coronavirus antiviral potential

Coronaviruses have caused multiple epidemics in the past two decades, in addition to the current COVID-19 pandemic that is severely damaging global health and the economy. Coronaviruses employ between 20 and 30 proteins to carry out their viral replication cycle, including infection, immune evasion, and replication. Among these, nonstructural protein 16 (Nsp16), a 2′-O-methyltransferase, plays an essential role in immune evasion. Nsp16 achieves this by mimicking its human homolog, CMTr1, which methylates mRNA to enhance translation efficiency and distinguish self from other. Unlike human CMTr1, Nsp16 requires a binding partner, Nsp10, to activate its enzymatic activity. The requirement of this binding partner presents two questions that we investigate in this manuscript. First, how does Nsp10 activate Nsp16? Although experimentally derived structures of the active Nsp16/Nsp10 complex exist, structures of inactive, monomeric Nsp16 have yet to be solved. Therefore, it is unclear how Nsp10 activates Nsp16. Using over 1 ms of molecular dynamics simulations of both Nsp16 and its complex with Nsp10, we investigate how the presence of Nsp10 shifts Nsp16’s conformational ensemble to activate it. Second, guided by this activation mechanism and Markov state models, we investigate whether Nsp16 adopts inactive structures with cryptic pockets that, if targeted with a small molecule, could inhibit Nsp16 by stabilizing its inactive state. After identifying such a pocket in SARS-CoV2 Nsp16, we show that this cryptic pocket also opens in SARS-CoV1 and MERS but not in human CMTr1. Therefore, it may be possible to develop pan-coronavirus antivirals that target this cryptic pocket.

Insight into the direct interaction of Na+ with NhaA and mechanistic implications

Na+/H+ antiporters comprise a family of membrane proteins evolutionarily conserved in all kingdoms of life that are essential in cellular ion homeostasis. While several human homologues have long been drug targets, NhaA of Escherichia coli has become the paradigm for this class of secondary active transporters as NhaA crystals provided insight in the structure of this molecular machine. However, structural data revealing the composition of the binding site for Na+ (or its surrogate Li+) is missing, representing a bottleneck in our understanding of the correlation between the structure and function of NhaA. Here, by adapting the scintillation proximity assay (SPA) for direct determination of Na+ binding to NhaA, we revealed that (i) NhaA is well adapted as the main antiporter for Na+ homeostasis in Escherichia coli and possibly in other bacteria as the cytoplasmic Na+ concentration is similar to the Na+ binding affinity of NhaA, (ii) experimental conditions affect NhaA-mediated cation binding, (iii) in addition to Na+ and Li+, the halide Tl+ interacts with NhaA, (iv) whereas acidic pH inhibits maximum binding of Na+ to NhaA, partial Na+ binding by NhaA is independent of the pH, an important novel insight into the effect of pH on NhaA cation binding.

SUMO-Modification of Human Nrf2 at K110 and K533 Regulates Its Nucleocytoplasmic Localization, Stability and Transcriptional Activity.

Background/Aims:Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that binds to the antioxidant response element(s) (ARE) in target gene promoters, enabling oxidatively stressed cells to respond in order to restore redox homeostasis. Post-translational modifications (PTMs) that mediate activation of Nrf2, in the cytosol and its release from Keap1, have been extensively studied but PTMs that impact its biology after activation are beginning to emerge. In this regard, PTMs like acetylation, phosphorylation, ubiquitination and sumoylation contribute towards the Nrf2 subcellular localization, and its transactivation function. We previously demonstrated that Nrf2 traffics to the promyelocytic leukemia-nuclear bodies (PML-NB), where it is a target for modification by small ubiquitin-like modifier (SUMO) proteins (sumoylation), but the site(s) for SUMO conjugation have not been determined. In this study, we aim to identify SUMO-2 conjugation site(s) and explore the impact, sumoylation of the site(s) have on Nrf2 stability, nuclear localization and transcriptional activation of its target gene expression upon oxidative stress.Methods:The putative SUMO-binding sites in Nrf2 for human isoform1 (NP_006155.2) and mouse homolog (NP_035032.1) were identified using a computer-based SUMO-predictive software (SUMOplot™). Site-directed mutagenesis, immunoblot analysis, and ARE-mediated reporter gene assays were used to assess the impact of sumoylation on these site(s) in vitro. Effect of mutation of these sumoylation sites of Nrf2 on expression of Heme Oxygenase1 (HO-1) was determined in HEK293T cell.Results:Eight putative sumoylation sites were identified by SUMOplot™ analysis. Out of the eight predicted sites only one 532LKDE535 of human (h) and its homologous 524LKDE527 of mouse (m) Nrf2, exactly matches the SUMO-binding consensus motif. The other high probability SUMO-acceptor site identified was residue K110, in the motifs 109PKSD112 and 109PKQD112 of human and mouse Nrf2, respectively. Mutational analysis of putative sumoylation sites (human (h)/mouse (m) K110, hK533 and mK525) showed that these residues are needed for SUMO-2 conjugation, nuclear localization and ARE driven transcription of reporter genes and the endogenous HO-1 expression by Nrf2. These residues also stabilized Nrf2, as evident from shorter half-lives of the mutant protein compared to wild-type Nrf2.Conclusion:Our findings indicate that SUMO-2 mediated sumoylation of K110 and K533 in human Nrf2 regulates in part its transcriptional activity by enhancing its stabilization and nuclear localization.

Derlin Rhomboid Pseudoproteases Employ Substrate Engagement and Lipid Distortion Function for Retrotranslocation of ER Multi-Spanning Membrane Substrates

Nearly one-third of proteins are initially targeted to the endoplasmic reticulum (ER) membrane where they are correctly folded, assembled, and then delivered to their final cellular destinations. In order to prevent the accumulation of misfolded membrane proteins, ER associated degradation (ERAD) moves these clients from the ER membrane to the cytosol; a process known as retrotranslocation. Our recent work in S. cerevisiae has revealed a derlin rhomboid pseudoprotease, Dfm1, is involved in the retrotranslocation of ubiquitinated ERAD membrane substrates. In this study, we sought to understand the mechanism associated with Dfm1’s actions and found that Dfm1’s conserved rhomboid residues are critical for membrane protein retrotranslocation. Specifically, we identified several retrotranslocation-deficient Loop 1 mutants that display impaired binding to membrane substrates. Furthermore, Dfm1 has retained the lipid thinning functions of its rhomboid protease predecessors to facilitate in the removal of ER membrane substrates. We find this substrate engagement and lipid thinning feature is conserved in its human homolog, Derlin-1. Utilizing interaction studies and molecular dynamics simulations, this work reveals that rhomboid pseudoprotease derlins employ novel mechanisms of substrate engagement and lipid thinning for catalyzing extraction of multi-spanning membrane substrates.

Naegleria fowleri: Protein structures to facilitate drug discovery for the deadly, pathogenic free-living amoeba

Naegleria fowleri is a pathogenic, thermophilic, free-living amoeba which causes primary amebic meningoencephalitis (PAM). Penetrating the olfactory mucosa, the brain-eating amoeba travels along the olfactory nerves, burrowing through the cribriform plate to its destination: the brain’s frontal lobes. The amoeba thrives in warm, freshwater environments, with peak infection rates in the summer months and has a mortality rate of approximately 97%. A major contributor to the pathogen’s high mortality is the lack of sensitivity of N. fowleri to current drug therapies, even in the face of combination-drug therapy. To enable rational drug discovery and design efforts we have pursued protein production and crystallography-based structure determination efforts for likely drug targets from N. fowleri. The genes were selected if they had homology to drug targets listed in Drug Bank or were nominated by primary investigators engaged in N. fowleri research. In 2017, 178 N. fowleri protein targets were queued to the Seattle Structural Genomics Center of Infectious Disease (SSGCID) pipeline, and to date 89 soluble recombinant proteins and 19 unique target structures have been produced. Many of the new protein structures are potential drug targets and contain structural differences compared to their human homologs, which could allow for the development of pathogen-specific inhibitors. Five of the structures were analyzed in more detail, and four of five show promise that selective inhibitors of the active site could be found. The 19 solved crystal structures build a foundation for future work in combating this devastating disease by encouraging further investigation to stimulate drug discovery for this neglected pathogen.

Hydrogen sulfide treatment at the late growth stage of Saccharomyces cerevisiae extends chronological lifespan.

Previous studies demonstrated that lifelong treatment with a slow H2S releasing donor extends yeast chronological lifespan (CLS), but it is not clear when the action of H2S benefits to CLS during yeast growth. Here, we show that short H2S treatments by using NaHS as a fast H2S releasing donor at 96 hours after inoculation extended yeast CLS while NaHS treatments earlier than 72 hours after inoculation failed to do so. To reveal the mechanism, we analyzed the transcriptome of yeast cells with or without the early and late NaHS treatments. We found that both treatments had similar effects on pathways related to CLS regulation. Follow-up qPCR and ROS analyses suggest that altered expression of some antioxidant genes by the early NaHS treatments were not stable enough to benefit CLS. Moreover, transcriptome data also indicated that some genes were regulated differently by the early and late H2S treatment. Specifically, we found that the expression of YPK2, a human SGK2 homolog and also a key regulator of the yeast cell wall synthesis, was significantly altered by the late NaHS treatment but not altered by the early NaHS treatment. Finally, the key role of YPK2 in CLS regulation by H2S is revealed by CLS data showing that the late NaHS treatment did not enhance the CLS of a ypk2 knockout mutant. This study sheds light on the molecular mechanism of CLS extension induced by H2S, and for the first time addresses the importance of H2S treatment timing for lifespan extension.

From Observed Action Identity to Social Affordances.

Others' observed actions cause continuously changing retinal images, making it challenging to build neural representations of action identity. The monkey anterior intraparietal area (AIP) and its putative human homologue (phAIP) host neurons selective for observed manipulative actions (OMAs). The neuronal activity of both AIP and phAIP allows a stable readout of OMA identity across visual formats, but human neurons exhibit greater invariance and generalize from observed actions to action verbs. These properties stem from the convergence in AIP of superior temporal signals concerning: (i) observed body movements; and (ii) the changes in the body-object relationship. We propose that evolutionarily preserved mechanisms underlie the specification of observed-actions identity and the selection of motor responses afforded by them, thereby promoting social behavior.

Lower visual field preference for the visuomotor control of limb movements in the human dorsomedial parietal cortex

Visual cues coming from the lower visual field (VF) play an important role in the visual guidance of upper and lower limb movements. A recently described region situated in the dorsomedial parietal cortex, area hPEc (Pitzalis et al. in NeuroImage 202:116092, 2019), might have a role in integrating visually derived information with somatomotor signals to guide limb interaction with the environment. In macaque, it has been demonstrated that PEc receives visual information mostly from the lower visual field but, to date, there has been no systematic investigation of VF preference in the newly defined human homologue of macaque area PEc (hPEc). Here we examined the VF preferences of hPEc while participants performed a visuomotor task implying spatially directed delayed eye-, hand- and foot-movements towards different spatial locations within the VF. By analyzing data as a function of the different target locations towards which upcoming movements were planned (and then executed), we observed the presence of asymmetry in the vertical dimension of VF in area hPEc, being this area more strongly activated by limb movements directed towards visual targets located in the lower compared to the upper VF. This result confirms the view, first advanced in macaque monkey, that PEc is involved in processing visual information to guide body interaction with the external environment, including locomotion. We also observed a contralateral dominance for the lower VF preference in the foot selective somatomotor cortex anterior to hPEc. This result might reflect the role of this cortex (which includes areas PE and S-I) in providing highly topographically organized signals, likely useful to achieve an appropriate foot posture during locomotion.

Sorting nexin Mdm1/SNX14 regulates nucleolar dynamics at the NVJ after TORC1 inactivation

The degradation of nucleolar proteins – nucleophagy – is elicited by nutrient starvation or the inactivation of target of rapamycin complex 1 (TORC1) protein kinase in budding yeast. Prior to nucleophagy, nucleolar proteins migrate to the nucleus–vacuole junction (NVJ), where micronucleophagy occurs, whereas rDNA (rRNA gene) repeat regions are condensed and escape towards NVJ-distal sites. This suggests that the NVJ controls nucleolar dynamics from outside of the nucleus after TORC1 inactivation, but its molecular mechanism is unclear. Here, we show that sorting nexin (SNX) Mdm1, an inter-organelle tethering protein at the NVJ, mediates TORC1 inactivation-induced nucleolar dynamics. Furthermore, Mdm1 was required for proper nucleophagic degradation of nucleolar proteins after TORC1 inactivation, where it was dispensable for the induction of nucleophagic flux itself. This indicated that nucleophagy and nucleolar dynamics are independently regulated by TORC1 inactivation. Finally, Mdm1 was critical for survival during nutrient starvation conditions. Mutations of SNX14, a human Mdm1 homolog, cause neurodevelopmental disorders. This study provides a novel insight into relationship between sorting nexin-mediated microautophagy and neurodevelopmental disorders.

Evolutionary Divergence in DNA Damage Responses among Fungi.

Cell cycle checkpoints and DNA repair pathways contribute to maintaining genome integrity and are thought to be evolutionarily ancient and broadly conserved. For example, in the yeast Saccharomyces cerevisiae and humans, DNA damage induces activation of a checkpoint effector kinase, Rad53p (human homolog Chk2), to promote cell cycle arrest and transcription of DNA repair genes. However, recent studies have revealed variation in the DNA damage response networks of some fungi. For example, Shor et al. (mBio 11:e03044-20, 2020, https://doi.org/10.1128/mBio.03044-20) demonstrate that in comparison to S. cerevisiae, the fungal pathogen Candida glabrata has reduced activation of Rad53p in response to DNA damage. Consequently, some downstream targets that contribute to S. cerevisiae genome maintenance, such as DNA polymerases, are transcriptionally downregulated in C. glabrata Downregulation of genome maintenance genes likely contributes to higher rates of mitotic failure and cell death in C. glabrata This and other recent findings highlight evolutionary diversity in eukaryotic DNA damage responses.

Role of lipid metabolism and systemic inflammation in the development of atherosclerosis in animal models

Systemic inflammation makes a significant contribution to the pathogenesis of atherosclerosis and has been the subject of numerous studies. Works aiming to analyze the mechanisms of atherosclerosis development often include experiments on animals. A primary task of such research is the characterization, justification, and selection of an adequate model.
 Aim. To evaluate the peculiarities of lipid metabolism and systemic inflammation in chronic obstructive pulmonary disease (COPD) in the development of atherosclerosis in animal models.
 Materials and Methods. Analyses of cross-links between species-specific peculiarities of lipid metabolism and the immune response, as well as a bioinformatic analysis of differences in Toll-like receptor 4 (TLR4) in mice, rats, and rabbits in comparison with its human homolog, were carried out. A search for and analysis of the amino acid sequences of human, mouse, rat, and rabbit TLR4 was performed in the International database GenBank of National Center of Biotechnical Information and in The Universal Protein Resource (UniProt) database. Multiple alignments of the TLR4 amino acid sequences were implemented in the Clustal Omega program, version 1.2.4. Reconstruction and visualization of molecular phylogenetic trees were performed using the MEGA7 program according to the Neighbor-Joining and Maximum Parsimony methods.
 Results. Species-specific differences of the peculiarities of lipid metabolism and the innate immune response in humans, mice, and rabbits were shown that must be taken into account in analyses of study results.
 Conclusion.Disorders in lipid metabolism and systemic inflammation mediated by the innate immune system participating in the pathogenesis of atherosclerosis in COPD possess species-specific differences that should be taken into account in analyses of study results.

Cex1 is a component of the COPI intracellular trafficking machinery.

ABSTRACTCOPI (coatomer complex I) coated vesicles are involved in Golgi-to-ER and intra-Golgi trafficking pathways, and mediate retrieval of ER resident proteins. Functions and components of the COPI-mediated trafficking pathways, beyond the canonical set of Sec/Arf proteins, are constantly increasing in number and complexity. In mammalian cells, GORAB, SCYL1 and SCYL3 proteins regulate Golgi morphology and protein glycosylation in concert with the COPI machinery. Here, we show that Cex1, homologous to the mammalian SCYL proteins, is a component of the yeast COPI machinery, by interacting with Sec27, Sec28 and Sec33 (Ret1/Cop1) proteins of the COPI coat. Cex1 was initially reported to mediate channeling of aminoacylated tRNA outside of the nucleus. Our data show that Cex1 localizes at membrane compartments, on structures positive for the Sec33 α-COP subunit. Moreover, the Wbp1 protein required for N-glycosylation and interacting via its di-lysine motif with the Sec27 β′-COP subunit is mis-targeted in cex1Δ deletion mutant cells. Our data point to the possibility of developing Cex1 yeast-based models to study neurodegenerative disorders linked to pathogenic mutations of its human homologue SCYL1.

Mutagenesis of a Lotus japonicus GSK3β/Shaggy-like kinase reveals functionally conserved regulatory residues

The glycogen synthase kinases 3 family (GSK3s/SKs; serine/threonine protein kinases) is conserved throughout eukaryotic evolution from yeast to plants and mammals. We studied a plant SK kinase from Lotus japonicus (LjSK1), previously implicated in nodule development, by enzyme kinetics and mutagenesis studies to compare it to mammalian homologues. Using a phosphorylated peptide as substrate, LjSK1 displays optimum kinase activity at pH 8.0 and 20 °C following Michaelis-Menten kinetics with Km and Vmax values of 48.2 μM and 111.6 nmol/min/mg, respectively, for ATP. Mutation of critical residues, as inferred by sequence comparison to the human homologue GSK3β and molecular modeling, showed a conserved role for Lys167, while residues conferring substrate specificity in the human enzyme are not as significant in modulating LjSK1 substrate specificity. Mutagenesis studies also indicate a regulation mechanism for LjSK1 via proteolysis since removal of a 98 residue long N-terminal segment increases its catalytic efficiency by almost two-fold. In addition, we evaluated the alteration of LjSK1 kinase activity in planta, by overexpressing the mutant variants in hairy-roots and a phenotype in nodulation and lateral root development was verified.

The Vps13 Family of Lipid Transporters and Its Role at Membrane Contact Sites.

The conserved VPS13 proteins constitute a new family of lipid transporters at membrane contact sites. These large proteins are suspected to bridge membranes and form a direct channel for lipid transport between organelles. Mutations in the 4 human homologs (VPS13A–D) are associated with a number of neurological disorders, but little is known about their precise functions or the relevant contact sites affected in disease. In contrast, yeast has a single Vps13 protein which is recruited to multiple organelles and contact sites. The yeast model system has proved useful for studying the function of Vps13 at different organelles and identifying the localization determinants responsible for its membrane targeting. In this review we describe recent advances in our understanding of VPS13 proteins with a focus on yeast research.

The Role of Hypoxia-Induced Mitogenic Factor in Organ-Specific Inflammation in the Lung and Liver: Key Concepts and Gaps in Knowledge Regarding Molecular Mechanisms of Acute or Immune-Mediated Liver Injury

Hypoxia-induced mitogenic factor (HIMF), which is also known as resistin-like molecule α (RELM-α), found in inflammatory zone 1 (FIZZ1), or resistin-like alpha (retlna), is a cysteine-rich secretory protein and cytokine. HIMF has been investigated in the lung as a mediator of pulmonary fibrosis, inflammation and as a marker for alternatively activated macrophages. Although these macrophages have been found to have a role in acute liver injury and acetaminophen toxicity, few studies have investigated the role of HIMF in acute or immune-mediated liver injury. The aim of this focused review is to analyze the literature and examine the effects of HIMF and its human homolog in organ-specific inflammation in the lung and liver. We followed the guidelines set by PRISMA in constructing this review. The relevant checklist items from PRISMA were included. Items related to meta-analysis were excluded because there were no randomized controlled clinical trials. We found that HIMF was increased in most models of acute liver injury and reduced damage from acetaminophen-induced liver injury. We also found strong evidence for HIMF as a marker for alternatively activated macrophages. Our overall risk of bias assessment of all studies included revealed that 80% of manuscripts demonstrated some concerns in the randomization process. We also demonstrated some concerns (54.1%) and high risk (45.9%) of bias in the selection of the reported results. The need for randomization and reduction of bias in the reported results was similarly detected in the studies that focused on HIMF and the liver. In conclusion, we propose that HIMF could be utilized as a marker for M2 macrophages in immune-mediated liver injury. However, we also detected the need for randomized clinical trials and additional experimental and human prospective studies in order to fully comprehend the role of HIMF in acute or immune-mediated liver injury.

Somatic and de novo Germline Variants of MEDs in Human Neural Tube Defects.

BackgroundNeural tube defects (NTDs) are among the most common and severe congenital defects in humans. Their genetic etiology is complex and remains poorly understood. The Mediator complex (MED) plays a vital role in neural tube development in animal models. However, no studies have yet examined the role of its human homolog in the etiology of NTDs.MethodsIn this study, 48 pairs of neural lesion site and umbilical cord tissues from NTD and 21 case-parent trios were involved in screening for NTD-related somatic and germline de novo variants. A series of functional cell assays were performed. We generated a Med12 p.Arg1784Cys knock-in mouse using CRISPR/Cas9 technology to validate the human findings.ResultsOne somatic variant, MED12 p.Arg1782Cys, was identified in the lesion site tissue from an NTD fetus. This variant was absent in any other normal tissue from different germ layers of the same case. In 21 case-parent trios, one de novo stop-gain variant, MED13L p.Arg1760∗, was identified. Cellular functional studies showed that MED12 p.Arg1782Cys decreased MED12 protein level and affected the regulation of MED12 on the canonical-WNT signaling pathway. The Med12 p.Arg1784Cys knock-in mouse exhibited exencephaly and spina bifida.ConclusionThese findings provide strong evidence that functional variants of MED genes are associated with the etiology of some NTDs. We demonstrated a potentially important role for somatic variants in the occurrence of NTDs. Our study is the first study in which an NTD-related variant identified in humans was validated in mice using CRISPR/Cas9 technology.