Divalent Cation Transporter Research Articles

Roles of Divalent-Cation Transporter Genes mntB and mntC of Beer Spoilage Bacteria in Resisting Hop Bitter Compound Iso-α-Acid

This study aimed to address the roles of divalent-cation transporter genes of Lactobacillus casei 2-9-5 in resisting hop bitter compound iso-α-acid. To determine the underlying mechanisms for Lactobacillus casei strains to resist iso-α-acid, RNA sequencing was performed for La. casei 2-9-5 and W2-9-5, which was an isolate of La. casei W2-9-5 derivative with a relatively weaker iso-α-acid resistance ability, in 0.012% iso-α-acid solution. Results indicated that its resistance of iso-α-acid was closely related to transmembrane transport, membrane composition and oxidative phosphorylation. Compared with La. casei W2-9-5, the expressions of the mntA, mntB and mntC genes were found to be significantly up-regulated in La. casei 2-9-5 and confirmed by RT-qPCR analysis. To examine the roles of these genes, recombinant plasmids harboring these genes were constructed and expressed in model bacteria Lactococcus lactis NZ3900. Relative to controls, the recombinant Lc. lactis containing mntB and mntC genes grew better under iso-α-acid pressure. Furthermore, the intracellular Mn2+ concentration in La. casei 2-9-5 was much higher than that in La. casei W2-9-5. Together, the data support that manganese transporter genes mntB and mntC are involved in the iso-α-acid resistance ability of La. casei strains through enhancement of intracellular Mn2+ concentrations.

Novel variant in the CNNM2 gene associated with dominant hypomagnesemia.

The maintenance of magnesium (Mg2+) homeostasis is essential for human life. The Cystathionine-β-synthase (CBS)-pair domain divalent metal cation transport mediators (CNNMs) have been described to be involved in maintaining Mg2+ homeostasis. Among these CNNMs, CNNM2 is expressed in the basolateral membrane of the kidney tubules where it is involved in Mg2+ reabsorption. A total of four patients, two of them with a suspected disorder of calcium metabolism, and two patients with a clinical diagnosis of primary tubulopathy were screened for mutations by Next-Generation Sequencing (NGS). We found one novel likely pathogenic variant in the heterozygous state (c.2384C>A; p.(Ser795*)) in the CNNM2 gene in a family with a suspected disorder of calcium metabolism. In this family, hypomagnesemia was indirectly discovered. Moreover, we observed three novel variants of uncertain significance in heterozygous state in the other three patients (c.557G>C; p.(Ser186Thr), c.778A>T; p.(Ile260Phe), and c.1003G>A; p.(Asp335Asn)). Our study shows the utility of Next-Generation Sequencing in unravelling the genetic origin of rare diseases. In clinical practice, serum Mg2+ should be determined in calcium and PTH-related disorders.

The metal binding site composition of the cation diffusion facilitator protein MamM cytoplasmic domain impacts its metal responsivity

The cation diffusion facilitator (CDF) is a conserved family of divalent d-block metal cation transporters that extrude these cations selectively from the cytoplasm. CDF proteins are composed of two domains: the transmembrane domain, through which the cations are transported, and a regulatory cytoplasmic C-terminal domain (CTD). It was recently shown that the CTD of the CDF protein MamM from magnetotactic bacteria has a role in metal selectivity, as binding of different metal cations exhibits distinctive affinities and conformations. It is yet unclear whether the composition of the CTD binding sites can impact metal selectivity and if we can manipulate the CTD to response to other non-native metals in CDF proteins. Here we performed a mutational study of the model protein MamM CTD, where we exchanged the native metal binding residues with different metal-binding amino acids. Using X-ray crystallography and Trp-fluorescence spectrometry, we studied the impact of these mutations on the CTD conformation in the presence of non-native metals. Our results reveal that the incorporation of such mutations alters the domain response to metals in vitro, as mutant forms of the CTD bind metals differently in terms of the composition of the binding sites and the CTD conformation. Therefore, the results demonstrate the direct influence of the CTD binding site composition on CDF proteins structure and hence, function, and constitute a first step for rational design of MamM for transporting different metals in vivo.

Immunohistochemistry Localisation of CNNM4 Protein during Tooth Murine Amelogenesis

Objectives: Homo sapiens cyclin and CBS domain divalent metal cation transport mediator 4 (CNNM4) is a protein that is suggested to play an important role in Mg2+ ion transport. Mutations in this gene are associated with Jalili syndrome which consists of cone-rod dystrophy and enamel defects. These results highlighted the importance of the CNNM4 during enamel formation, so our work aimed to study the localisation of CNNM4 during different stages of lower incisor and first molar murine amelogenesis. Material and Methods: Adult incisors (n: 16) of 4 week mice (C57BL) and pups at PNO, PN4 and PN14 were used to study Cnnm4 expression in different stages of mouse incisor and first molar respectively. The pups were obtained from time mated pregnant mice. Results: In lower incisor, Cnnm4 was detected in ameloblast only in the maturation stage while in the secretory stage the signals was detected in a very specific way to stratum intermedium (SI). While in lower first molar, Cnnm4 was localised in inner enamel epithelium (pre-ameloblast) and stratum intermedium of the enamel organ at early bell stage (pre-secretory stage) while stellate reticulum and outer enamel epithelium were negative. In addition to that during the secretory stage, Cnnm4 signal was detected in secretory ameloblast under the cusp tip. Dental papilla, odontoblasts and dental follicles were appeared completely negative in both lower incisor and first molar. Conclusion: CNNM4 has a specific expression pattern in enamel forming cells during different stages of teeth development in mice amelogenesis.

Genome-Wide Identification of Metal Tolerance Protein Genes in Populus trichocarpa and Their Roles in Response to Various Heavy Metal Stresses.

Metal tolerance proteins (MTPs) are plant divalent cation transporters that play important roles in plant metal tolerance and homeostasis. Poplar is an ideal candidate for the phytoremediation of heavy metals because of its numerous beneficial attributes. However, the definitive phylogeny and heavy metal transport mechanisms of the MTP family in poplar remain unknown. Here, 22 MTP genes in P. trichocarpa were identified and classified into three major clusters and seven groups according to phylogenetic relationships. An evolutionary analysis suggested that PtrMTP genes had undergone gene expansion through tandem or segmental duplication events. Moreover, all PtrMTPs were predicted to localize in the vacuole and/or cell membrane, and contained typical structural features of the MTP family, cation efflux domain. The temporal and spatial expression pattern analysis results indicated the involvement of PtrMTP genes in poplar developmental control. Under heavy metal stress, most of PtrMTP genes were induced by at least two metal ions in roots, stems or leaves. In addition, PtrMTP8.1, PtrMTP9 and PtrMTP10.4 displayed the ability of Mn transport in yeast cells, and PtrMTP6 could transport Co, Fe and Mn. These findings will provide an important foundation to elucidate the biological functions of PtrMTP genes, and especially their role in regulating heavy metal tolerance in poplar.

Cadmium exposure enhances cell migration and invasion through modulated TRPM7 channel expression

Cadmium is a xenobiotic involved in neoplastic transformation. Cadmium enters the cells through divalent cation transporters including the Transient Receptor Potential Melastatin-related 7 (TRPM7) which is known to be involved in cancer cell fate. This work aimed to study the role of TRPM7 in neoplastic transformation induced by cadmium exposure in non-cancer epithelial cells. Non-cancer epithelial cells were chronically exposed to low-dose of cadmium. TRPM7 expression and function were studied by Western-Blot, Patch-Clamp and calcium and magnesium imaging. Finally, cell migration and invasion were studied by Boyden chamber assays. Chronic cadmium exposure induced TRPM7 overexpression and increased the membrane currents (P < 0.001). Cells exposed to cadmium had higher intracellular calcium and magnesium levels (P < 0.05). TRPM7 silencing restored calcium levels but strongly decreased intracellular magnesium concentration (P < 0.001). Moreover, cadmium exposure enhanced both cell migration and invasion, but TRPM7 silencing strongly decreased these features (P < 0.001). Furthermore, mammary epithelial cells exposed to cadmium became rounded and had less cell-to-cell junctions. Cadmium exposure decreased epithelial markers while the mesenchymal ones were increased. Importantly, TRPM7 silencing was able to reverse these phenotypic modifications (P < 0.05). To summarize, our data show that chronic cadmium exposure enhanced TRPM7 expression and activity in non-cancer epithelial cells. TRPM7 overexpression induced intracellular magnesium increase and stimulated cell migration and invasion. These neoplastic properties could be linked to a TRPM7-dependent epithelial-to-mesenchymal transition reprogramming in cell exposed to cadmium. These findings provide new insights into the regulation of cell fates by cadmium exposure.

Tacrolimus-induced hypomagnesemia and hypercalciuria requires FKBP12 suggesting a role for calcineurin.

Calcineurin inhibitors (CNIs) are immunosuppressive drugs used to prevent graft rejection after organ transplant. Common side effects include renal magnesium wasting and hypomagnesemia, which may contribute to new‐onset diabetes mellitus, and hypercalciuria, which may contribute to post‐transplant osteoporosis. Previous work suggested that CNIs reduce the abundance of key divalent cation transport proteins, expressed along the distal convoluted tubule, causing renal magnesium and calcium wasting. It has not been clear, however, whether these effects are specific for the distal convoluted tubule, and whether these represent off‐target toxic drug effects, or result from inhibition of calcineurin. The CNI tacrolimus can inhibit calcineurin only when it binds with the immunophilin, FKBP12; we previously generated mice in which FKBP12 could be deleted along the nephron, to test whether calcineurin inhibition is involved, these mice are normal at baseline. Here, we confirmed that tacrolimus‐treated control mice developed hypomagnesemia and urinary calcium wasting, with decreased protein and mRNA abundance of key magnesium and calcium transport proteins (NCX‐1 and Calbindin‐D28k). However, qPCR also showed decreased mRNA expression of NCX‐1 and Calbindin‐D28k, and TRPM6. In contrast, KS‐FKBP12−/− mice treated with tacrolimus were completely protected from these effects. These results indicate that tacrolimus affects calcium and magnesium transport along the distal convoluted tubule and strongly suggests that inhibition of the phosphatase, calcineurin, is directly involved.

Mg2+-ATP Sensing in CNNM, a Putative Magnesium Transporter

The family of cystathionine-β-synthase (CBS)-pair domain divalent metal cation transport mediators (CNNMs) is composed of four integral membrane proteins associated with Mg2+ transport. Structurally, CNNMs contain large cytosolic regions composed of a CBS-pair and a cyclic nucleotide-binding homology (CNBH) domain. How these regulate Mg2+ transport activity is unknown. Here, we determined the crystal structures of cytosolic fragments in two conformations: Mg2+-ATP-analog bound and ligand free. The structures reveal open and closed conformations with functionally important contacts not observed in structures of the individual domains. We also identified a second Mg2+-binding region in the CBS-pair domain and a different dimerization interface for the CNBH domain. Analytical ultracentrifugation and isothermal titration calorimetry experiments revealed a tight correlation between Mg2+-ATP binding and protein dimerization. Mutations that blocked either function prevented cellular Mg2+ efflux activity. The results suggest Mg2+ efflux is regulated by conformational changes associated with Mg2+-ATP binding to CNNM CBS-pair domains.

Structural Insights into the Intracellular Region of the Human Magnesium Transport Mediator CNNM4.

The four member family of “Cyclin and Cystathionine β-synthase (CBS) domain divalent metal cation transport mediators”, CNNMs, are the least-studied mammalian magnesium transport mediators. CNNM4 is abundant in the brain and the intestinal tract, and its abnormal activity causes Jalili Syndrome. Recent findings show that suppression of CNNM4 in mice promotes malignant progression of intestinal polyps and is linked to infertility. The association of CNNM4 with phosphatases of the regenerating liver, PRLs, abrogates its Mg2+-efflux capacity, thus resulting in an increased intracellular Mg2+ concentration that favors tumor growth. Here we present the crystal structures of the two independent intracellular domains of human CNNM4, i.e., the Bateman module and the cyclic nucleotide binding-like domain (cNMP). We also derive a model structure for the full intracellular region in the absence and presence of MgATP and the oncogenic interacting partner, PRL-1. We find that only the Bateman module interacts with ATP and Mg2+, at non-overlapping sites facilitating their positive cooperativity. Furthermore, both domains dimerize autonomously, where the cNMP domain dimer forms a rigid cleft to restrict the Mg2+ induced sliding of the inserting CBS1 motives of the Bateman module, from a twisted to a flat disk shaped dimer.

A novel pathogenic missense variant in CNNM4 underlying Jalili syndrome: Insights from molecular dynamics simulations

BackgroundJalili syndrome (JS) is a rare cone‐rod dystrophy (CRD) associated with amelogenesis imperfecta (AI). The first clinical presentation of JS patients was published in 1988 by Jalili and Smith. Pathogenic mutations in the Cyclin and CBS Domain Divalent Metal Cation Transport Mediator 4 (CNNM4) magnesium transporter protein have been reported as the leading cause of this anomaly.MethodsIn the present study, a clinical and genetic investigation was performed in a consanguineous family of Pakistani origin, showing characteristic features of JS. Sanger sequencing was successfully used to identify the causative variant in CNNM4. Molecular dynamics (MD) simulations were performed to study the effect of amino acid change over CNNM4 protein.ResultsSequence analysis of CNNM4 revealed a novel missense variant (c.1220G>T, p.Arg407Leu) in exon‐1 encoding cystathionine‐β‐synthase (CBS) domain. To comprehend the mutational consequences in the structure, the mutant p.Arg407Leu was modeled together with a previously reported variant (c.1484C>T, p.Thr495Ile) in the same domain. Additionally, docking analysis deciphered the binding mode of the adenosine triphosphate (ATP) cofactor. Furthermore, 60ns MD simulations were carried out on wild type (p.Arg407/p.Thr495) and mutants (p.Arg407Leu/p.Thr495Ile) to understand the structural and energetic changes in protein structure and its dynamic behavior. An evident conformational shift of ATP in the binding site was observed in simulated mutants disrupting the native ATP‐binding mode.ConclusionThe novel identified variant in CNNM4 is the first report from the Pakistani population. Overall, the study is valuable and may give a novel insight into metal transport in visual function and biomineralization.

Genome-Wide Identification, Comprehensive Gene Feature, Evolution, and Expression Analysis of Plant Metal Tolerance Proteins in Tobacco Under Heavy Metal Toxicity.

Plant metal tolerance proteins (MTPs) comprise a family of membrane divalent cation transporters that play essential roles in plant mineral nutrition maintenance and heavy metal stresses resistance. However, the evolutionary relationships and biological functions of MTP family in tobacco remain unclear. In the present study, 26, 13, and 12 MTPs in three main Nicotiana species (N. tabacum, N. sylvestris, and N. tomentosiformis) were identified and designated, respectively. The phylogenetic relationships, gene structures, chromosome distributions, conserved motifs, and domains of NtMTPs were systematic analyzed. According to the phylogenetic features, 26 NtMTPs were classified into three major substrate-specific groups that were Zn-cation diffusion facilitators (CDFs), Zn/Fe-CDFs, and Mn-CDFs, and seven primary groups (1, 5, 6, 7, 8, 9, and 12). All of the NtMTPs contained a modified signature sequence and the cation_efflux domain, whereas some of them also harbored the ZT_dimer. Evolutionary analysis showed that NtMTP family of N. tabacum originated from its parental genome of N. sylvestris and N. tomentosiformis, and further underwent gene loss and expanded via one segmental duplication event. Moreover, the prediction of cis-acting elements (CREs) and the microRNA target sites of NtMTP genes suggested the diverse and complex regulatory mechanisms that control NtMTPs gene expression. Expression profile analysis derived from transcriptome data and quantitative real-time reverse transcription-PCR (qRT-PCR) analysis showed that the tissue expression patterns of NtMTPs in the same group were similar but varied among groups. Besides, under heavy metal toxicity, NtMTP genes exhibited various responses in either tobacco leaves or roots. 19 and 15 NtMTPs were found to response to at least one metal ion treatment in leaves and roots, respectively. In addition, NtMTP8.1, NtMTP8.4, and NtMTP11.1 exhibited Mn transport abilities in yeast cells. These results provided a perspective on the evolution of MTP genes in tobacco and were helpful for further functional characterization of NtMTP genes.

The β-Lactamase Gene Profile and a Plasmid-Carrying Multiple Heavy Metal Resistance Genes of Enterobacter cloacae.

In this work, by high-throughput sequencing, antibiotic resistance genes, including class A (bla CTX-M, bla Z, bla TEM, bla VEB, bla KLUC, and bla SFO), class C (bla SHV, bla DHA, bla MIR, bla AZECL-29, and bla ACT), and class D (bla OXA) β-lactamase genes, were identified among the pooled genomic DNA from 212 clinical Enterobacter cloacae isolates. Six bla MIR-positive E. cloacae strains were identified, and pulsed-field gel electrophoresis (PFGE) showed that these strains were not clonally related. The complete genome of the bla MIR -positive strain (Y546) consisted of both a chromosome (4.78 Mb) and a large plasmid pY546 (208.74 kb). The extended-spectrum β-lactamases (ESBLs) (bla SHV-12 and bla CTX-M-9a) and AmpC (bla MIR) were encoded on the chromosome, and the pY546 plasmid contained several clusters of genes conferring resistance to metals, such as copper (pco), arsenic (ars), tellurite (ter), and tetrathionate (ttr), and genes encoding many divalent cation transporter proteins. The comparative genomic analyses of the whole plasmid sequence and of the heavy metal resistance gene-encoding regions revealed that the plasmid sequences of Klebsiella pneumoniae (such as pKPN-332, pKPN-3967, and pKPN-262) shared the highest similarity with those of pY546. It may be concluded that a variety of β-lactamase genes present in E. cloacae which confer resistance to β-lactam antibiotics and the emergence of plasmids carrying heavy metal resistance genes in clinical isolates are alarming and need further surveillance.

PDK2 induces cisplatin-resistance in lung adenocarcinoma via transcriptional regulation of CNNM3

Recurrence of lung adenocarcinoma has become one of the most frequent causes of major cancer incidence and mortality worldwide according to its frequently gained resistance to chemotherapies. In this study, we identified a poorly-studied kinase pyruvate dehydrogenase kinase isoform 2 (PDK2) as the most up-regulated kinase encoding gene in Cisplatin resistant lung adenocarcinoma. Additionally, PDK2-dependent Cisplatin-resistance promotes tumour growth of lung adenocarcinoma both in vitro and in vivo. Clinically, PDK2 expression was up-regulated in lung adenocarcinoma and was correlated to the poor prognosis of lung cancer patients. Mechanically, PDK2 promoted cell growth and Cisplatin-resistance of lung adenocarcinoma via transcriptional regulation of cyclin and CBS domain divalent metal cation transport mediator 3 (CNNM3), indicating that PDK2-CNNM3 signalling axis could be a potential therapeutic target for Cisplatin-resistant lung adenocarcinoma.

Quantification of Mg2+, Ca2+ and H+ transport by the gastrointestinal tract of the goldfish, Carassius auratus, using the Scanning Ion-selective Electrode Technique (SIET).

An in vitro gut-sac technique and the scanning ion-selective electrode technique (SIET) were used to characterize Mg2+, Ca2+, and H+ transport at both the mucosal and serosal surfaces of non-everted and everted gastrointestinal tissues obtained from Carassius auratus. As part of the study, two magnesium ionophores were compared (II vs. VI). Unfed animals displayed uniform transport of all ions along the intestine. Feeding resulted in elevated Mg2+ and Ca2+ transport when the gut lumen contained chyme however, under symmetrical conditions this increased transport rate was absent. Furthermore, zonation of divalent cation transport was present for both Ca2+ and Mg2+ under non-symmetrical conditions while the zonation remained for Ca2+ alone under symmetrical conditions. High dietary Mg2+ decreased absorption and induced secretion of Mg2+ in the posterior intestine. Uptake kinetics in the esophagus suggest large diffusive and/or convective components based on a linear relationship between Mg2+ transport and concentration and lack of inhibition by ouabain, an inhibitor of Na+-K+-ATPase. In contrast, kinetics in the rectum were suggestive of a low affinity, saturable carrier-mediated pathway. A decrease in Mg2+ and Ca2+ transport was observed in the posterior intestine (both at the mucosal and serosal surfaces) in response to ouabain. This impact was greatest for Ca2+ transport and when applied to the mucosal fluid and measured in everted preparations. In contrast a putative Mg2+ transport inhibitor, cobalt(III)hexamine-chloride, did not affect Mg2+ transport. This is the first study to use SIET approaches to study ion transport in the gut of teleost fish. This is also the first study to provide characterization of Mg2+ transport in the gut of C. auratus. Due to the limited selectivity of Magnesium ionophore II, subsequent studies of tissues bathed in physiological saline should be made using Magnesium Ionophore VI.

SLC11A1 is expressed in the human placenta across multiple gestational ages

The human placenta functions as an innate immune barrier to prevent fetal infection. However, the molecular mechanisms accounting for placental resistance to pathogens are currently poorly understood. The solute carrier family 11 member 1 (SLC11A1) is a divalent cation transporter expressed primarily by macrophages and neutrophils that is essential for controlling infections by intracellular pathogens such as Salmonella, Leishmania and Mycobacteria. This report demonstrates that SLC11A1 is expressed in the syncytiotrophoblast of the human placenta at multiple gestational ages. These results suggest that SLC11A1 may play a role in blocking productive placental infections by certain intracellular pathogens.

The homologous Arabidopsis MRS2/MGT/CorA-type Mg2+ channels, AtMRS2-10 and AtMRS2-1 exhibit different aluminum transport activity

Magnesium (Mg2+) plays a critical role in many physiological processes. The AtMRS2/MGT family, which consists of nine Arabidopsis genes (and two pseudo-genes) belongs to a eukaryotic subset of the CorA superfamily of divalent cation transporters. AtMRS2-10 and AtMRS2-1 possess the signature GlyMetAsn sequence conserved in the CorA superfamily; however, they have low sequence conservation with CorA. Direct measurement using the fluorescent dye mag-fura-2 revealed that reconstituted AtMRS2-10 and AtMRS2-1 mediated rapid Mg2+ uptake into proteoliposomes. The rapid Mg2+ uptake through AtMRS2-10 was inhibited by aluminum. An assay using the Al-sensitive dye morin indicated Al uptake into the proteoliposomes through AtMRS2-10. AtMRS2-10 also exhibited Ni2+ transport activity but almost no Co2+ transport activity. The rapid Mg2+ uptake through AtMRS2-1 was not inhibited by aluminum. Al uptake into the proteoliposomes through AtMRS2-1 was not observed. The functional complementation assay in Escherichia coli strain TM2 showed that AtMRS2-1 was capable of mediating Mg2+ uptake. Heterologous expression using the E. coli mutant cells also showed that the E. coli cells expressing AtMRS2-1 was more resistant to aluminum than the E. coli cells expressing AtMRS2-10. The results suggested that AtMRS2-10 transported Al into the E. coli cells, and then the transported Al inhibited the growth of E. coli. AtMRS2-1 has been localized to the Arabidopsis tonoplast, indicating that AtMRS2-1 is exposed to much higher concentration of aluminum than AtMRS2-10. Under the conditions, it may be required that the Mg2+ transport of AtMRS2-1 is insensitive to Al inhibition, and AtMRS2-1 is impermeable to Al.

In utero gene expression in the Slc39a8(neo/neo) knockdown mouse

Slc39a8 encodes ZIP8, a divalent cation/bicarbonate symporter expressed in pluripotent mouse embryonic stem cells, and therefore ubiquitous in adult tissues; ZIP8 influxes Zn2+, Mn2+ and Fe2+. Slc39a8(neo/neo) knockdown mice exhibit 10–15% of wild-type ZIP8 mRNA and protein levels, and show pleiotropic phenotype of stunted growth, neonatal lethality, multi-organ dysmorphogenesis, and dysregulated hematopoiesis manifested as severe anemia. Herein we performed RNA-seq analysis of gestational day (GD)13.5 yolk sac and placenta, and GD16.5 liver, kidney, lung, heart and cerebellum, comparing Slc39a8(neo/neo) with Slc39a8(+/+) wild-type. Meta-data analysis of differentially-expressed genes revealed 29 unique genes from all tissues — having enriched GO categories associated with hematopoiesis and hypoxia and KEGG categories of complement, response to infection, and coagulation cascade — consistent with dysregulated hematopoietic stem cell fate. Based on transcription factor (TF) profiles in the JASPAR database, and searching for TF-binding sites enriched by Pscan, we identified numerous genes encoding zinc-finger and other TFs associated with hematopoietic stem cell functions. We conclude that, in this mouse model, deficient ZIP8-mediated divalent cation transport affects zinc-finger (e.g. GATA proteins) and other TFs interacting with GATA proteins (e.g. TAL1), predominantly in yolk sac. These data strongly support the phenotype of dysmorphogenesis and anemia seen in Slc39a8(neo/neo) mice in utero.

Loss of conserved ubiquitylation sites in conserved proteins during human evolution.

Ubiquitylation of lysine residues in proteins serves a pivotal role in the efficient removal of misfolded or unused proteins and in the control of various regulatory pathways by monitoring protein activity that may lead to protein degradation. The loss of ubiquitylated lysines may affect the ubiquitin‑mediated regulatory network and result in the emergence of novel phenotypes. The present study analyzed mouse ubiquitylation data and orthologous proteins from 62mammals to identify 193 conserved ubiquitylation sites from 169 proteins that were lost in the Euarchonta lineage leading to humans. A total of 8 proteins, including betaine homocysteine S‑methyltransferase, clin and CBS domain divalent metal cation transport mediator 3, ribosome‑binding protein 1 and solute carrier family 37 member 4, lost 1 conserved lysine residue, which was ubiquitylated in the mouse ortholog, following the human‑chimpanzee divergence. A total of 17 of the lost ubiquitylated lysines are also known to be modified by acetylation and/or succinylation in mice. In 8cases, a novel lysine evolved at positions flanking the lost conserved lysine residues, potentially as a method of compensation. We hypothesize that the loss of ubiquitylation sites during evolution may lead to the development of advantageous phenotypes, which are then fixed by selection. The ancestral ubiquitylation sites identified in the present study may be a useful resource for investigating the association between loss of ubiquitylation sites and the emergence of novel phenotypes during evolution towards modern humans.

Transcriptome sequencing analysis reveals the effect of combinative treatment with low‑intensity pulsed ultrasound and magnesium ions on hFOB1.19 human osteoblast cells.

Biodegradable magnesium (Mg) materials are considered ideal as osteosynthesis implants. However, clinical application has proven complex. This is primarily associated with the issue of reducing the extent of implant degradation to a range acceptable for the human body, while simultaneously enhancing osteogenesis or osteoinduction. In the present study, a combination of Mg ions and low-intensity pulsed ultrasound (LIPUS) treatment was applied in hFOB 1.19 human osteoblast cells as a potential strategy to resolve this issue. A total of 7,314 differentially expressed genes (DEGs) and 826 shared DEGs in hFOB1.19 osteoblast cells were identified by microarray analysis following treatment with Mg and/or LIPUS. Gene Ontology analysis demonstrated that among cells treated with a combination of Mg and LIPUS, DEGs were significantly enriched in various functional annotations, including ‘wound healing’, ‘transforming growth factor beta receptor signaling pathway’, ‘transcription, DNA-templated’, ‘receptor complex’, ‘nucleus’, ‘SMAD protein complex’, ‘DNA binding’, ‘metal ion binding’ and ‘GTPase activator activity’. Notably, the transforming growth factor (TGF)-β, mitogen-activated protein kinase (MAPK) and tumor necrosis factor (TNF) signaling pathways were preferentially overrepresented in the Mg and LIPUS combination group, which was subsequently confirmed by reverse transcription-quantitative polymerase chain reaction. Furthermore, genes involved in osteoblast mineralization promotion, including bone morphogenetic protein 6, noggin, bone morphogenetic protein receptor (BMPR)1A, BMPR2 and SMAD 5/8, were significantly upregulated following combination treatment compared with the control group. Genes involved in the promotion of migration, including c-Jun N-terminal kinase, doublecortin, paxillin and Jun proto-oncogene AP-1 transcription factor subunit, were also upregulated in the combination treatment group compared with the control group. The DEG data were supported by morphological observations of the osteoblasts using alizarin red S staining and wound healing assays, which indicated that Mg and LIPUS combinative treatment had a synergistic effect on osteoblast mineralization and migration. Additionally, the combined treatment significantly upregulated metal transporter genes associated with Mg entry, including ATPase Na+/K+-transporting subunit α1, cyclin and CBS domain divalent metal cation transport mediator 2, K+ voltage-gated channel subfamily J member 14, transient receptor potential cation channel (TRP) subfamily M member 7 and TRP subfamily V member 2. In summary, the findings of the present study revealed that combined stimulation with Mg and LIPUS may exhibit a synergistic effect on human osteoblast bone formation through the TGF-β, MAPK and TNF signaling pathways, while also facilitating Mg influx. The present study demonstrated the potential of combinative LIPUS and Mg treatment as a novel therapeutic strategy for enhancing the osteoinduction, biocompatibility and biosafety of biodegradable Mg implants.

Sequence characterization, structural analysis, SNP detection and expression profiling of SLC11A1 gene in Indian goats

The Solute Carrier Family 11 Member A1 (SLC11A1) is a candidate gene, encodes a divalent cation transporter, SLC11A1 protein, localizes in the phagolysosome membrane in macrophages, essential for immunity and resistance to intracellular pathogens. The indigenous goats of Kerala are known for disease resistance and adaptability, but the underlying genetic causes are to be unraveled. This study comprised of characterization of the caprine SLC11A1 gene, to detect SNPs and compare the relative abundance of SLC11A1 mRNA by quantitative-PCR in native goats (Attappady Black and Malabari) and crossbreds. The 1702 bp cDNA of SLC11A1 comprised of 15 exons, an ORF of 1647 bp, encoding a protein of 548 amino acids. SLC11A1 amino acid sequence was 83–99% identical to that of other species. SLC11A1 peptide contains 10 serine and 2 threonine phosphorylation loci and 12 transmembrane domains. The study indicated the presence of four synonymous and three non-synonymous mutations (p.Ile132Thr; p.Phe294Leu; p.Gln541His). The amino acid substitution p.Ile132Thr was deleterious and p.Phe294Leu and p.Gln541His were neutral. p.Phe294Leu was located in the 7th transmembrane helix. The relative abundance of the SLC11A1 mRNA in leukocytes in Malabari and Attappady Black were significantly higher than crossbreds (P < 0.01). The significantly higher SLC11A1 expression in native goats indicates higher disease resistance than crossbreds, gives an insight into the necessity of conservation of native breeds, important role of SLC11A1 in disease resistance. The detected SNPs would benefit the use of this gene as a candidate gene for disease resistance in goat breeding programs.