The Vacuolar Transporter OsNRAMP2 Mediates Fe Remobilization During Germination and Affects Cd Distribution to Rice Grain

Background and aims Iron (Fe) deficiency in plants is a common problem affecting agricultural production. Cadmium (Cd) is a toxic metal that can be taken up and transported within plants by transporters for divalent metals including Fe(II). The present study aims to investigate the functions of OsNRAMP2 (Natural Resistance-Associated Macrophage Protein 2) in the remobilization and distribution of Fe and Cd in rice. Methods The expression pattern of OsNRAMP2 was determined by quantitative real-time PCR and pOsNRAMP2:GUS assay. Knockout mutants of OsNRAMP2 were generated by using CRISPR/Cas9 gene editing. Localization of Fe in the vacuolar globoids of germinating seeds was imaged by high-resolution transmission electron microscopy coupled with energy-dispersive X-ray spectroscopy. Distributions of Fe and Cd between different plant tissues were investigated in hydroponic and soil pot experiments. Results OsNRAMP2 was mainly expressed in the embryo of germinating seeds, roots, leaf sheaths and leaf blades. OsNRAMP2 was localized at the tonoplast. Knockout of OsNRAMP2 delayed seed germination and produced chlorotic seedling leaves. Remobilization of Fe stored in the protein storage vacuoles in the scutellum of germinating seeds was restricted in osnramp2 mutants compared with wild type. Expression of genes related to Fe uptake was enhanced in the seedlings of osnramp2 mutants. Knockout of OsNRAMP2 significantly decreased the distribution of Cd, but not Fe, from leaves and straws to rice grains. Conclusions OsNRAMP2 plays an important role in remobilizing vacuolar Fe during seed germination and affects translocation of Cd from vegetative tissues to rice grains.

Manganese is a Physiologically Relevant TORC1 Activator in Yeast and Mammals.

The essential biometal manganese (Mn) functions as a cofactor for several enzymatic activities that are critical for the prevention of human diseases. Whether intracellular Mn levels may also modulate signaling events has so far remained largely unexplored. The target of rapamycin complex 1 (TORC1, mTORC1 in mammals) is a conserved protein kinase complex that requires metal co-factors to phosphorylate its downstream effectors as part of a central, homeostatic process that coordinates cell growth and metabolism in response to nutrient and/or growth factor availability. Using genetic and biochemical approaches, we show here that TORC1 activity is exquisitely sensitive to stimulation by Mn both in vivo and in vitro. Mn-mediated control of TORC1 depends on Smf1 and Smf2, two members of the family of natural resistance-associated macrophage protein (NRAMP) metal ion transporters, the turnover of which is subjected to feedback control by TORC1 activity. Notably, increased Mn levels and consequent activation of TORC1 cause retrograde dysregulation and antagonize the rapamycin-induced gene expression and autophagy programs in yeast. Because Mn also activates mTORC1 signaling in aminoacid starved human cells, our data indicate that intracellular Mn levels may constitute an evolutionary conserved physiological cue that modulates eukaryotic TORC1/mTORC1 signaling. Our findings therefore reveal a hitherto elusive connection between intracellular Mn levels, mTORC1 activity, and human diseases.

Genome-Wide Identification and Expression Analysis of the NRAMP Family Genes in Tea Plant (Camellia sinensis)

The natural resistant-associated macrophage protein (NRAMP) is a kind of integral membrane transporter which could function on a wide range of divalent metal ions in plants. Little is known about the NRAMP family in Camellia sinensis. In this study, 11 NRAMP genes were identified from the tea plant genome. Phylogenetic analysis showed that the 11 CsNRAMP proteins were split into two groups. The proteins of group 1 contained the conserved motif 6 (GQSSTxTG), while most proteins in group 2 (excepting CsNRAMP7 and CsNRAMP10) contained the conserved residues of motif 6 and motif 2 (GQFIMxGFLxLxxKKW). The number of amino acids in coding regions of 11 CsNRAMP genes ranged from 279–1373, and they contained 3–12 transmembrane domains. Quantitative RT-PCR analysis showed that G1 genes, CsNRAMP3, CsNRAMP4, and CsNRAMP5, were extraordinarily expressed in roots, while G2 genes showed higher expression levels in the stems and leaves. The expression levels of CsNRAMPs in roots and leaves were detected to assess their responses to Pb treatment. The results indicated that CsNRAMPs were differentially regulated, and they might play a role in Pb transportation of tea plant. Subcellular localization assay demonstrated that CsNRAMP2 and CsNRAMP5 fused proteins were localized in the plasma membrane. Overall, this systematic analysis of the CsNRAMP family could provide primary information for further studies on the functional roles of CsNRAMPs in divalent metal transportation in tea plants.

Natural resistance-associated macrophage protein-1 gene polymorphisms and genetic susceptibility to pulmonary tuberculosis in sudanese patients

Genetic susceptibility to tuberculosis has been investigated worldwide, however a few studies conducted in Sudanese patients. Polymorphisms in NRAMP1 have shown an important association with susceptibility to tuberculosis and progression to active disease. However, whether there is an association of these polymorphisms with pulmonary tuberculosis among Sudanese patients is not investigated yet. The aim of this study was to determine the association of NRAMP1 polymorphisms with development of pulmonary tuberculosis in Sudanese subjects with pulmonary tuberculosis. A case-controls study (100 in each arm) was conducted at Al-Shaab Teaching Hospital in Khartoum, Sudan during the period of March through September 2017. Genotyping for NRAMP1 (rs3731865) INTR4 and (rs17235409) D543N was done by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP).There was no significant difference in the mean± SD of the age and body mass index (BMI) between the cases (tuberculosis) and the controls [37.7 ± 4.1 vs. 35.9 ± 3.9 years; P=0.702] and [24.6 ± 1.9 vs. 24.7 ± 2.1; P=0.444], respectively. None of the genotype frequencies investigated in the two polymorphisms were associated with the development of pulmonary tuberculosis. There was a significant association with the development of tuberculosis with illiteracy P=<0.001. Gene polymorphisms of NRAMP1 (rs3731865) INTR4 and (rs17235409) D543N were not associated with pulmonary tuberculosis development in this setting. Further study is needed with larger sample size and different design and settings. Increase public awareness about tuberculosis infections and specially in those who are illiterate.

Recombinant apoptosis inhibitor of macrophage protein reduces delayed graft function in a murine model of kidney transplantation

Reperfusion injury following cold and warm ischemia (IRI) is unavoidable during kidney transplantation and contributes to delayed graft function (DGF) and premature graft loss. Death of tubular epithelial cells (TECs) by necrosis during IRI releases pro-inflammatory mediators (e.g. HMGB1), propagating further inflammation (necroinflammation) and tissue damage. Kidney Injury Molecule-1 (KIM-1) is a phagocytic receptor upregulated on proximal TECs during acute kidney injury. We have previously shown that renal KIM-1 protects the graft against transplant associated IRI by enabling TECs to clear apoptotic and necrotic cells, and that recognition of necrotic cells by KIM-1 is augmented in the presence of the opsonin, apoptosis inhibitor of macrophages (AIM). Here, we tested whether recombinant AIM (rAIM) could be used to mitigate transplant associated IRI. We administered rAIM or vehicle control to nephrectomised B6 mice transplanted with a single B6 donor kidney. Compared to grafts in vehicle-treated recipients, grafts from rAIM-treated mice exhibited significantly less renal dysfunction, tubular cell death, tissue damage, tubular obstruction, as well as local and systemic inflammation. Both mouse and human rAIM enhanced the clearance of necrotic cells by murine and human TECs, respectively in vitro. These data support testing of rAIM as a potential therapeutic agent to reduce DGF following kidney transplantation.

The Tonoplast-Localized Transporter OsNRAMP2 is involved in Fe Homeostasis and affects Seed Germination in rice

Vacuolar storage of iron (Fe) is important for Fe homeostasis in plants. When sufficient, the excess Fe could be stored in vacuoles for remobilization in case of Fe deficiency. Although the mechanism of Fe remobilization from vacuoles is critical for crop development under low Fe stress, the transporters that mediate vacuolar Fe translocation into the cytosol in rice remains unknown. Here, we showed that under higher Fe 2+ concentrations, the Δccc1 yeast mutant transformed with rice natural resistance-associated macrophage protein 2 (OsNRAMP2) became more sensitive to Fe toxicity. In rice protoplasts and transgenic plants expressing Pro35S: OsNRAMP2-GFP, OsNRAMP2 was localized to tonoplast. Vacuolar Fe contents in osnramp2 knockdown lines were higher than in the wild-type, while the growth of osnramp2 knockdown plants was significantly influenced by Fe deficiency. Furthermore, the germination of osnramp2 knockdown plants was arrested. Inversely, the vacuolar Fe contents of Pro35S: OsNRAMP2-GFP lines were significantly lower than in the wild-type, and overexpression of OsNRAMP2 increased shoot biomass under Fe deficiency. Taken together, we propose that OsNRAMP2 transports Fe from the vacuole to the cytosol and plays a pivotal role in seed germination.

Association ofNRAMP1-INT4 gene with Susceptibility to Tuberculosis in the Sudanese Population: A Case-Control Study

BackgroundSudan is a Sub-Saharan African country with a high prevalence rate of Tuberculosis. Natural Resistant Associated Macrophage Protein 1 (NRAMP1) plays a potential role in the development of immunity against TB, and it has a critical role in disease resistance. The aim of the study was to evaluate the association ofNRAMP1polymorphism at intron4 (INT4) region with susceptibility to TB infection.MethodsDemographic, clinical and microbiological data were collected from 150 participants and investigated using designed questionnaire. The genotyping ofNRAMP1-INT4 polymorphism was performed in 60 TB-infected patients and 50 healthy control using Polymerase chain reaction and restriction fragment-length polymorphism method (PCR-RFLP).ResultsAmong cases (60%) were males, only (3.3%) were vaccinated. The most reported risk factors were tobacco smoking (17%), diabetes (10%), alcohol consumption (2%) and corticosteroid therapy intakes (1%). Pulmonary TB was detected in 67% of the patients, 24% had pulmonary/MDR and 9% had extra-pulmonary TB. The frequency of wild G allele was significantly higher in cases compared with healthy control subjects (P-value <0.0001). Also, a significant association was observed between the heterozygosity forNRAMP1-INT4 variant and resistance to TB infection (P-value 0.001, OR= 4.83, 95%CI 1.96∼11.88). Homozygotes mutant INT4 (C/C) genotype was not detected in both cases and controls.ConclusionstheNRAMP1-INT4 polymorphism may serve as marker of unidentified genetic factors that may play a critical role in host immunity to TB in the Sudanese population. Further studies with large sample size are recommended to determine population-specific genetic associations with TB susceptibility in order to guide TB therapy and prophylaxis in a population-specific manner.

Human Genetic Variation Influences Enteric Fever Progression

In the 21st century, enteric fever is still causing a significant number of mortalities, especially in high-risk regions of the world. Genetic studies involving the genome and transcriptome have revealed a broad set of candidate genetic polymorphisms associated with susceptibility to and the severity of enteric fever. This review attempted to explain and discuss the past and the most recent findings on human genetic variants affecting the progression of Salmonella typhoidal species infection, particularly toll-like receptor (TLR) 4, TLR5, interleukin (IL-) 4, natural resistance-associated macrophage protein 1 (NRAMP1), VAC14, PARK2/PACRG, cystic fibrosis transmembrane conductance regulator (CFTR), major-histocompatibility-complex (MHC) class II and class III. These polymorphisms on disease susceptibility or progression in patients could be related to multiple mechanisms in eliminating both intracellular and extracellular Salmonella typhoidal species. Here, we also highlighted the limitations in the studies reported, which led to inconclusive results in association studies. Nevertheless, the knowledge obtained through this review may shed some light on the development of risk prediction tools, novel therapies as well as strategies towards developing a personalised typhoid vaccine.