Here, we show that CNNM4 is the basolaterally located Mg2+ extrusion molecule. CNNM4 is strongly expressed in intestinal epithelia and localizes to their basolateral membrane. CNNM4-knockout mice showed hypomagnesemia due to the intestinal malabsorption of magnesium, suggesting its role in Mg2+ extrusion to the inner parts of body. Imaging analyses revealed that CNNM4 can extrude Mg2+ by exchanging intracellular Mg2+ with extracellular Na+. Furthermore, CNNM4 mutations cause Jalili syndrome, characterized by recessive amelogenesis imperfecta with cone-rod dystrophy. CNNM4-knockout mice showed defective amelogenesis, and CNNM4 again localizes to the basolateral membrane of ameloblasts, the enamel-forming epithelial cells. Missense point mutations associated with the disease abolish the Mg2+ extrusion activity. These results demonstrate the crucial importance of Mg2+ extrusion by CNNM4 in organismal and topical regulation of magnesium.Transcellular Mg 2+ transport across epithelia, involving both entry from one side and extrusion from the other side, is important. Previous studies have revealed the role of Mg2+-permeable channel protein in Mg2+ entry into the epithelial cells. However, the identity of proteins involved in Mg2+ extrusion to the inner parts of body has remained unknown. Mice genetically engineered not to express CNNM4, which localizes to the epithelial membrane facing to the inner parts of body, show hypomagnesemia due to the defect in magnesium absorption. Functional analyses using culture cells directly reveal that CNNM4 can extrude intracellular Mg2+ to the outside of cells. These results indicate that CNNM4 mediates transcellular Mg2+ transport across the intestinal epithelia. Furthermore, we also show that these CNNM4-lacking mice also have a defect in amelogenesis, which is consistent with the disease symptoms of Jalili syndrome that is known to be caused by mutations in the CNNM4 gene.Magnesium is an essential element for living organisms. Its absorption occurs at the intestine through the barrier comprised of epithelial cells. In this process, transcellular Mg Genomic analyses of familial cases of hypomagnesemia have identified key molecules directly involved in these processes. CLDN16, encoding claudin-16/paracellin-1, and CLDN19, encoding claudin-19, are mutated in recessive familial hypomagnesemia with hypercalciuria and nephrocalcinosis 2+ between adjacent epithelial cells TRPM6 cause recessive hypomagnesemia with secondary hypocalcemia Magnesium is an essential element involved in a wide variety of biological activities. Homeostasis of the magnesium level is strictly regulated by intestinal absorption and renal reabsorption, in which epithelia function as a barrier that permits selective and regulated transport of Mg2+-permeable ion channel that localizes to the apical membrane of epithelial cells in the intestine and kidney 2+ into cells, which is the first step in transcellular Mg2+ absorption across the epithelial barrier, another major Mg2+ transport pathway. To accomplish Mg2+ absorption, epithelial cells need to extrude Mg2+ via their basolateral membrane by opposing the inward-oriented driving force on Mg2+ imposed by the electrical membrane potential. Such a transcellular Mg2+ transport mechanism, involving both apical entry and basolateral extrusion, is evolutionarily conserved from Caenorhabditis elegans2+ extrusion have not been identified.TRPM6 is a member of the transient receptor potential melastatin-related (TRPM) protein family and constitutes a MgCNNM genes and magnesium homeostasis. Several single nucleotide polymorphisms in CNNM genes are associated with the serum magnesium level CNNM2 cause familial dominant hypomagnesemia Salmonella, CorC, has been suggested to participate in Mg2+ efflux Xenopus oocytes showed voltage-dependent transport of several divalent cations, including Mg2+CNNM2 could restore the growth of a Mg2+-deficient Salmonella strain + current 2+ transport still remains unknown. Moreover, it has been reported that mutations in CNNM4 cause Jalili syndrome, which is characterized by recessive amelogenesis imperfecta (AI) and cone-rod dystrophy (CRD) Ancient conserved domain protein/cyclin M (CNNM) constitutes a family of 4 integral membrane proteins that possess an evolutionarily conserved but uncharacterized domain from bacteria 2+ absorption. Endogenous CNNM4 is highly expressed in the mature ameloblasts and intestinal epithelia, and localizes at their basolateral membrane. Functional analyses at the molecular and organismal levels revealed a common role for CNNM4 in mediating transcellular Mg2+ transport by basolateral Mg2+ extrusion.In this study, we generated CNNM4-knockout mice; these mice showed defects in amelogenesis and intestinal MgCNNM4 by homologous recombination  cell clone, which possesses the neomycin-resistance gene cassette inserted in the genomic region between the first and second exons of bination . Chimeriblotting  and routblotting . The genblotting  confirme protein . Both CN−/−CNNM4 mice showed no positive signals, thus confirming that the signal at the mucosal epithelia properly reflects the localization of endogenous CNNM4.Immunoblotting analyses of lysates obtained from various organs showed that CNNM4 is highly expressed in the small intestine and colon , consistTo precisely determine the subcellular localization of CNNM4, we also performed immunofluorescence microscopy. Low-magnification images confirmed the specific expression of CNNM4 in the mucosal epithelia . In the +/K+ ATPase , immediately beneath ZO-1.To further confirm the basolateral localization of CNNM4, we ectopically expressed CNNM4-FLAG in MDCK cells, which maintain a highly polarized epithelial character in culture. As shown in 2+ transporter, localizes to the basolateral membrane of the intestinal epithelia suggests the involvement of CNNM4 in the regulation of magnesium homeostasis. To explore this possibility, we analyzed the magnesium levels in −/−CNNM4 mice maintained on a normal diet (CLEA Rodent Diet CE-2 containing 0.34% magnesium). Magnesium quantitation, using the colorimetric reagent Xylidyl Blue-I, showed that −/−CNNM4 mice had a significantly lower serum magnesium concentration: an approximately 18% decrease was observed in comparison to +/+CNNM4 mice  to examine the levels of several major metal elements in serum. As shown in −/−CNNM4 mice, whereas the magnesium level was significantly reduced. We then observed mice fed a magnesium-deficient diet (containing 0.0027% magnesium) and found a significant increase in mortality in −/−CNNM4 mice , without a significant difference in the quantity of food ingested. These symptoms are very similar to those of the TRPM7-mutant mice, which have defects in intestinal magnesium absorption Magnesium homeostasis is regulated by the balance between intestinal absorption and renal excretion. The decrease in renal excretion can be considered to reflect a compensatory response to maintain magnesium levels during hypomagnesemia caused by intestinal malabsorption. To directly measure the effect on intestinal absorption, we analyzed the magnesium content in feces. As shown in 2+ extrusion. Other analyzed elements  showed no significant differences. We next performed imaging analyses with Magnesium Green, a fluorescent indicator for Mg2+. HEK293 cells transfected with CNNM4-FLAG were first loaded with Mg2+ by bathing them in a solution containing 40 mM Mg2+, which was then exchanged with a Mg2+-free solution to artificially promote Mg2+ extrusion. As shown in 2+ depletion, whereas only a very subtle decrease was observed in empty vector-transfected cells. Thus, CNNM4 is able to stimulate Mg2+ extrusion.To clarify the molecular function of CNNM4, we first examined the effect of CNNM4-overexpresion on the intracellular levels of major metal elements by using ICP-ES. As shown in 2+ to move inward into cells, and thus, energy supply is needed to actively extrude Mg2+ to the outside. Many proteins involved in active transport across the plasma membrane utilize the large electrochemical potential of Na+. To determine the importance of extracellular Na+ in Mg2+ extrusion, we first performed Mg2+ extrusion assays by replacing Na+ in the medium with another cation, N-methyl-D-glucamine (NMDG). In this case, Mg2+ extrusion was completely abolished (“−Na+” in 2+ depletion in the medium caused Mg2+ extrusion in CNNM4-expressing cells (Phase 1) and addition of 40 mM Mg2+ restored intracellular Mg2+ (Phase 2). In the absence of extracellular Na+, Mg2+ depletion did not induce Mg2+ extrusion (Phase 3), but restoration of Na+ instantaneously caused Mg2+ extrusion (Phase 4). Such tight coupling between the presence of extracellular Na+ and the occurrence of Mg2+ extrusion further supports the notion that CNNM4 stimulates Na+/Mg2+ exchange; this is also consistent with the sodium increase observed in CNNM4-expressing cells  hexammine (CoHex), which broadly inhibits channel-mediated Mg2+ influx 2+ recovery , CNNM4-expressing cells incorporated significant amount of Mg2+ even at 10 mM. Furthermore, we observed that even though the Mg2+ level in CNNM4-expressing cells was lower than that in the control cells before loading, it became much higher after the loading procedure with 10 mM Mg2+, 0 mM Na+ solution, and then returned to the basal level when extracellular Mg2+ was removed. These data strongly suggest the occurrence of the reverse action of CNNM4 and corroborate our notion that CNNM4 stimulates Na+/Mg2+ exchange.To determine whether the rapid restoration of intracellular Mgrecovery , suggest 78.1 mM . However+, as reported previously 2+ extrusion, we next performed simultaneous Mg2+ imaging and electrophysiological recording experiments. The exchange of the extracellular solution with an Mg2+-free solution stimulated rapid Mg2+ decrease without inducing significant electronic currents in CNNM4-expressing cells  and Mag-fura2, respectively. As shown in 2+ depletion from the extracellular medium induced not only the decrease of intracellular Mg2+ but also the increase of intracellular Na+. In addition, the molar ratio of increased Na+ and decreased Mg2+ was calculated to be 2.14∶1, which is roughly consistent with the electroneutral exchange of Na+ and Mg2+ (2∶1). To quantitatively assess the dependency of Mg2+ extrusion on the presence of extracellular Na+, we performed Mg2+ extrusion assays by changing the concentration of extracellular Na+. Extracellular Na+ accelerated Mg2+ extrusion in a dose-dependent manner, and the Hill coefficient was calculated to be 1.90, a value close to 2 . The low-magnification images showed that the thickness of the enamel layer in −/−CNNM4 mice was not so different from that in +/+CNNM4 mice  and smooth-ended (SA) ameloblasts, which can be discerned by ZO-1-staining −/− mice .2+ extrusion by CNNM4, we examined whether missense point mutations in CNNM4, which have been reported to occur in the patients of Jalili syndrome 2+ extrusion activity. We tested the effect of two different point mutations, viz., S200Y and L324P, both of which occur in the evolutionarily conserved DUF21 domain  analyses. To observe the retinal histology, we stained retinal sections from 2-month-old (young adult) −/−CNNM4 mice with toluidine blue. We found that the retinal layers were normal and no symptom of retinal degeneration was observed in the retina of −/−CNNM4 mice  are normal in the −/−CNNM4 retina .Another feature of Jalili syndrome is CRD, which is characterized with the degeneration of rod and cone photoreceptors in the retina −/− mice . We also− retina . Cone phRP3, one of causative genes of human hereditary retinal diseases −/−CNNM4 mice were performed. However, we did not observe any signs of histological abnormalities  is much faster than that by SLC41A1 (after ∼10 min) 2+ extrusion has not been reported in the previous studies characterizing the endogenous Mg2+ extrusion systems in non-intestinal cells 2+ extrusion molecule, which may have a specialized role in the intestinal epithelia. Magnesium absorption from the intestine is essential for magnesium homeostasis, and 100–150 mg magnesium is daily absorbed from the intestine in humans 2+ entry into the intestinal epithelial cells is mediated by apically localized Mg2+-permeable channels TRPM6/7 that can rapidly incorporate Mg2+2+ extrusion from the basolateral membrane is mediated by high capacity transporters, such as CNNM4, to achieve efficient transcellular Mg2+ transport through intestinal epithelia.In this study, we have shown that CNNM4 localizes to the basolateral membrane of epithelial cells and extrudes Mg−/−CNNM4 mice showed a defect in magnesium absorption, but were viable, without any significant observable phenotype when fed a normal diet. CNNM proteins comprise a family of 4 related proteins, CNNM1–4 −/−CNNM4 mice can be ascribed to the functional complementation by other CNNM family proteins. CNNM4 is expressed in the intestine, but not in the kidney, and thus, it will not affect renal reabsorption, the other key process in the regulation of magnesium homeostasis. The amount of magnesium reabsorbed from the glomerular filtrate is estimated to be about 10 times that absorbed from digested food. Therefore, the absence of CNNM4 in the kidney raises the next important question of what molecule is responsible for Mg2+ extrusion from distal convoluted tubule (DCT) cells in the kidney, where TRPM6 is expressed at the apical membrane and where transcellular Mg2+ transport occurs 2+ transport cooperatively with TRPM6. It should be noted here that SLC41A1 is also expressed in the DCT cells and its gene mutation causes nephronophthisis-related disorder CNNM4 is mutated in Jalili syndrome, which is characterized by recessive AI and CRD −/−CNNM4 mice showed no signs of abnormalities in the retinal tissue architecture and function , and received approval for this study from the institutional review board of Osaka University.CNNM4 by homologous recombination. The ES cells were used to generate germline chimeras that were bred with C57BL/6J females to generate CNNM4-knockout mice. Southern blot analyses were performed to confirm appropriate recombination. Genomic DNA of mice was digested with EcoRV and hybridized with the external or neo probes. Genotyping PCR was performed using the following primer set: 5′-TAACTGTTGGAAGGCTGAGG-3′ and 5′-AGGCAGGGGCTCCCTTTCAT-3′. Mice were maintained under standard specific pathogen-free conditions.We purchased an ES clone (ID: EPD0426_1_C08) from EUCOMM, in which the neomycin-resistant gene cassette had been inserted in the genomic region between the first and second exons of CNNM4 cDNA was purchased from Invitrogen (IMAGE: 30340626). Amino acid substituted mutants S200Y and L324P were generated with the QuickChange Site-Directed Mutagenesis Kit (Agilent). An anti-CNNM4 rabbit polyclonal antibody was raised in rabbits immunized with bacterially expressed His-CNNM4 proteins (amino acids 546–775) and purified with corresponding GST-tagged recombinant proteins. Anti-ZO-1 mouse monoclonal antibody was generated in the previous study +/K+ ATPase mouse monoclonal antibody (#05-369) and anti-M-opsin rabbit polyclonal antibody (AB5405) were purchased from Merck Millipore. Anti-FLAG rabbit polyclonal antibody (F7425) and anti-PKCα rabbit polyclonal antibody (P4334) were purchased from Sigma-Aldrich. Anti-Ctbp2 mouse monoclonal antibody (612044) was purchased from BD Biosciences. Anti-Rhodopsin (LB-5597) and anti-Calbindin (PC253L) rabbit polyclonal antibodies were purchased from LSL and Calbiochem, respectively. Anti-S-opsin goat polyclonal antibody (sc-14363) was purchased from Santa Cruz Biotechnology. Alexa Fluor 488-conjugated anti-rabbit IgG was purchased from Invitrogen and Sigma-Aldrich. Alexa Fluor 488-conjugated anti-mouse IgG was purchased from Sigma-Aldrich. Alexa Fluor 568-conjugated anti-mouse IgG, and rhodamine-labelled phalloidin were purchased from Invitrogen. Cy3-conjugated anti-rabbit, -goat and -guinea pig IgGs were purchased from Jackson ImmunoResearch Laboratories. Rhodamine-labeled PNA (RL1072) was purchased from Vector Laboratories.Human CNNM4 (Invitrogen), which target the following sequence: CNNM4-siRNA, 5′-GCGAGAGCAUGAAGCUGUAUGCACU-3′, were used. As control, we used siRNA representing a scrambled sequence of CNNM4-siRNA, 5′-GCGACGAAAGUGUCGGUAUCGAACU-3′.HEK293 cells and MDCK cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and antibiotics. Transient expression and knockdown were achieved using LipofectAmine2000 (Invitrogen) to transfect cells with plasmids or siRNAs according to the manufacturer's instruction. Plasmid constructs in the pCMV-Tag 4 vector (Agilent Technologies) were used for expression of CNNM4. For knockdown experiments, duplex siRNAs against human 2O2. After blocking with PBS containing 3% fetal bovine serum and 10% bovine serum albumin for 1 h at room temperature, specimens were incubated with the primary antibodies overnight at 4°C, followed by incubation with the peroxidase-conjugated secondary antibodies for 1 h at room temperature. Immunostaining was developed with diaminobenzidine and counterstained with Mayer's haematoxylin. The specimens were observed under a microscope . Differential Interference Contrast (DIC) images were collected using an inverted microscope .For intestine preparation, intestines were dissected from 2-month-old mice, embedded in OCT compound , frozen in liquid nitrogen, and then sectioned into at 10-µm sections using a cryostat (Leica). The sections were mounted on glass slides, air-dried, and fixed with phosphate-buffered saline (PBS) containing 4% paraformaldehyde (PFA) for 10 min at 4°C. For mandible preparation, 6-week-old mice were anesthetized and fixed by perfusion with PBS containing 4% PFA. Mandibles were dissected out, fixed with PBS containing 4% PFA for 12 h at 4°C, decalcified with 10% EDTA for 2 weeks, dehydrated with xylene through a graded ethanol series, and embedded in paraffin. Sections (4-µm thick) were cut using a microtome (Leica), and then mounted on glass slides. Slides were heat-treated in Pascal, a pressure chamber (Dako) and cooled at room temperature after deparaffinization and rehydration. Both frozen and paraffin-embedded sections were then incubated with PBS containing 0.3% H+/K+-ATPase, cells were permeabilized with 0.1% TritonX-100 for 5 min at room temperature. After blocking with PBS containing 3% fetal bovine serum and 10% bovine serum albumin (blocking buffer) for 1 h, cells were incubated for 12 h with the primary antibody diluted in blocking buffer. After 3 washes with PBS, cells were incubated for 30 min with the appropriate secondary antibodies diluted in blocking buffer. Cryosections of intestines and paraffin-embedded sections were prepared as described above. When stained for ZO-1, sections were permeabilized with ice-cold acetone for 3 min after fixation. Fixed sections were blocked and incubated with the primary and secondary antibodies as for cultured cells. After washing with PBS, coverglasses were mounted with Aqueous Mounting Medium PermaFluor (Thermo SCIENTIFIC) and observed with a confocal scanning laser microscope . The procedure of immunofluorescent analysis of retinas was described previously Cells cultured on coverglasses were washed with PBS and fixed with 1% formaldehyde for 15 min at room temperature. When stained for ZO-1, cells were permeabilized with 0.5% TritonX-100 in PBS for 10 min at room temperature. When stained for Na2 (photopic units) maximum intensity. 4 levels of stimulus intensities ranging from −4.0 to 1.0 log cd-s/m2 were used for the scotopic ERG recordings, and 4 levels of stimuli ranging from −0.5 to 1.0 log cd-s/m2 were used for the photopic ERGs. Animals were light adapted for 10 min before the photopic ERG recordings. 8 and 16 responses were averaged for photopic (−4.0 and −3.0 log) and all scotopic recordings, respectively.ERG responses were measured after overnight dark adaptation using PuREC system with LED electrodes (Mayo Corporation) g for 20 min at 4°C. Urine and feces samples were collected from 2-month-old mice by using metabolic cages (CLEA Japan). Feces were air-dried, incubated with 1 N nitric acid  overnight, and then centrifuged. The magnesium concentration of the supernatant was determined using Xylidyl Blue-I (Wako) according to the manufacturer's instructions.Mice were fed either a normal diet containing 0.34% magnesium  or a magnesium-deficient diet containing 0.0027% magnesium (CLEA Japan). Blood samples were obtained from 8-week-old mice. These were incubated at 4°C overnight, and serum was then collected by centrifugation at 1,000× Serum samples were mixed with HCl at a final concentration of 1% and incubated at 95°C for 2 h. Samples were then subjected to elementary analysis with ICPS-8100 (Shimadzu), according to the manufacturer's instructions. The mean of triplicate measurements was used to represent the result of a single sample. The results were normalized to total protein levels, which were determined by the Bradford method.2+-imaging analyses with Magnesium Green were performed as follows. HEK293 cells were incubated with Mg2+-loading buffer , including 2 µM Magnesium Green-AM (Invitrogen), for 45 min at 37°C. The cells were rinsed once with loading buffer and viewed using a microscope . Fluorescence was measured every 20 sec (excitation at 470–490 nm and emission at 505–545 nm) under the control of the Metamorph software (Molecular Devices). Then, the buffer was changed to −Mg2+ buffer (MgCl2 in the loading buffer was replaced with 60 mM NaCl), or to −Mg2+−Na+ buffer (NaCl in −Mg2+ buffer was replaced with NMDG-Cl). The data are presented as line plots (mean of 10 cells). After imaging analyses, cells were fixed with PBS containing 3.7% formaldehyde and subjected to immunofluorescence microscopy to confirm protein expression. Cobalt (III) hexammine was purchased from SIGMA.Mg2 until use. Patch-clamp experiments under the whole-cell configuration were performed according to Stuiver et al., 4, 10 mM HEPES (pH 7.35 adjusted with Tris). The intracellular solution was 120 mM NMDG, 120 mM 2-(N-morpholino)-ethanesulfonic acid hydrate, 2 mM MgSO4, 10 mM HEPES (pH 7.2 adjusted with H2SO4). All solutions were adjusted to 295–305 mOsm with sucrose.pIRES-HcRed plasmids 2+-imaging and electrophysiological recording experiments were performed with IX71 microscope (Olympus) equipped with iXon EM-CCD camera (Andor Technology) and a xenon lamp in Lambda DG-4 illumination system (Sutter Instrument). Borosilicate patch pipettes had resistances of 3–5 MΩ after filled with the intracellular solution containing 2 µM Magnesium Green . Cells were voltage clamped to −10 mV, and the imaging was started after the fluorescent intensities from the cell became stabilized (20–35 min after the establishment of the whole-cell configuration). The fluorescence was measured every 20 sec (excitation at 470–490 nm and emission at 505–545 nm). Mg2+-loading buffer and −Mg2+ buffer were used as extracellular solutions. The intracellular solution was 2 mM MgCl2, 2 mM NaCl, 5 mM EGTA, 140 mM KCl, 5 mM HEPES (pH 7.25 adjusted with KOH). All solutions were adjusted to 295–305 mOsm with sucrose.Simultaneous Mg2 until use. Mg2+ extrusion assays were performed with the abovementioned protocol, with following modifications. Cells were loaded with 2 µM Mag-fura2-AM or 3 µM SBFI-AM (Invitrogen) and viewed using the IX81 microscope (Olympus) equipped with ORCA-Flash 4.0 CMOS camera (Hamamatsu Photonics) and USH-1030L mercury lamp (Olympus). The fluorescence was measured every 20 sec , and −Mg2+ buffer with various Na+ concentrations (prepared by replacing NaCl with NMDG-Cl) was used to stimulate Mg2+ efflux.HEK293 cells were transfected with expression plasmids for CNNM4, and maintained in normal culture media plus 40 mM MgCl2+ and Na+  were determined from the following equation:1) to that with 370–390 nm excitation (F2) (R = F1/F2). Rmax: the maximum value of R. Rmin: the minimum value of R. Q: the ratio of the signal intensity with 370–390 nm excitation under minimum Mg2+ or Na+ concentration to the signal intensity with 370–390 nm excitation under maximum Mg2+ or Na+ concentration (F2min/F2max). Kd: 1.5 mM for Mag-fura2 min, Rmax, Fmin, Fmax were obtained after each experiment. For Mag-fura2, Rmin, Fmin were recorded by addition of 6 µM 4-Bromo-A23187 (Wako) and 10 mM EDTA, and Rmax, Fmax were recorded by incubating the cells under −Mg2+ buffer plus 6 µM 4-Bromo-A23187 and 50 mM MgCl2. For SBFI, Rmax, Fmax were recorded by incubating the cells under −Mg2+ buffer supplemented with 5 µM Gramicidin (Wako), and Rmin, Fmin were recorded by incubating the cells under the Na+-depleted buffer (a −Mg2+ buffer which NaCl is replaced with KCl) with 5 µM Gramicidin. The cells were fixed with PBS containing 3.7% formaldehyde after fluorescence measurement and subjected to immunofluorescence microscopy to confirm protein expression. Difference of [Na+]i and [Mg2+]i just after Mg2+ depletion (between time = 0 and 20 sec) was used to determine the initial velocity of Na+ influx (V0 (Na)) and Mg2+ efflux (V0 (Mg)), respectively. The ratio of CNNM4-dependent Na+ influx versus Mg2+ efflux was calculated as follows:max, KA, and Hill coefficient were determined by SigrafW software Intracellular concentrations of free Mg2+ loading assays were performed with the abovementioned protocol for ratiometric imaging, with following modifications. Cells were incubated in −Mg2+ buffer with 2 µM Mag-fura2-AM for 10 min, 37°C. The cells were once rinsed with −Mg2+ buffer and viewed using the same apparatuses. Then, the extracellular solution was changed to buffers with various Mg2+ and Na+ concentrations (buffer with low Na+ concentrations were prepared by replacing NaCl with NMDG-Cl) and incubated for 4 min to load Mg2+. Finally, the extracellular solution was changed to −Mg2+ buffer to stimulate Mg2+ efflux.HEK293 cells were transfected with expression plasmids for CNNM4, and maintained in normal culture media until use. MgMaxillae dissected from 2-month-old mice were fixed with 70% ethanol for 5 days, dehydrated in ascending alcohol series, and embedded in methyl methacrylate. After embedding, cutting specimen, and surface polishing, the samples were then coated with room-temperature ionic liquid (1-butyl-3-methylimidazolium tetrafluoroborate), which work as an electric conductor and enables the observation of biological specimen by an SEM Figure S1Characterization of the anti-CNNM4 antibody. Lysates of HEK293 cells transfected with CNNM4-FLAG or CNNM4-siRNA were subjected to immunoblotting analyses with the anti-CNNM4 antibody. The endogenous CNNM4 signal in HEK293 cells is indicated with an arrowhead.(PDF)Click here for additional data file.Figure S2Basolateral localization of ectopically expressed CNNM4 in MDCK epithelial cells. MDCK cells transfected with CNNM4-FLAG were subjected to immunofluorescence staining with the antibodies for FLAG (green) and ZO-1 (red) or Na+/K+ ATPase (red). Horizontal section images (X–Y), at the level of the apical and lateral membranes, and vertically reconstituted images (X–Z) are shown. Vertical sections were taken at the white lines indicated in the X–Y images. Bar, 10 µm.(PDF)Click here for additional data file.Figure S32+ influx in CNNM4-expressing cells. (A) HEK293 cells expressing CNNM4-FLAG were loaded with Magnesium Green and then subjected to time-lapse imaging analyses in the presence of 1 or 3 mM CoHex, under various extracellular solutions. The Mg2+ concentration in the extracellular solution is indicated. The means of relative fluorescence intensities of 10 cells are indicated. (B) HEK293 cells expressing CNNM4-FLAG were loaded with Mag-fura2, and then subjected to Mg2+ loading assays. The cells were incubated with extracellular solutions containing various concentrations of Mg2+ and Na+ during the indicated period (“Mg2+ loading”). The Mg2+ and Na+ concentrations in the extracellular solutions during the loading period, and the means of [Mg2+]i of 4 independent experiments (10 cells for each experiment) are indicated. See Characterization of Mg(PDF)Click here for additional data file.Figure S42+ (n = 5–6). (C) Current densities at −110 mV recorded either in the presence or absence of extracellular Mg2+. Data are presented as mean ± s.e.m. of 5–6 cells. p value was determined by Student's two-tailed t-test. n.s.: not significant. (D) HEK293 cells transfected with the indicated constructs were subjected to simultaneous Mg2+ imaging and electrophysiological recording experiments. The extracellular solution was changed from Mg2+-containing to an Mg2+-free solution at the time point indicated by arrowheads. Means of relative fluorescence intensities and current densities (at −10 mV) of 4–6 cells are indicated. (E) Decreased fluorescence intensities and induced current densities were observed during the period between the arrows in (D). Data are presented as mean ± s.e.m. of 4–6 cells. p values were determined by Student's two-tailed t-test. ***p<0.001, n.s.: not significant.Electrophysiological recordings. (A) HEK293 cells transfected with the indicated expression constructs were subjected to electrophysiological recordings under whole-cell configuration. The cells were voltage-clamped between −120 mV and 70 mV in steps of 10 mV, and the representative traces of each cell are indicated. (B) Average I–V relationship of control (green), CNNM2- (blue), or CNNM4- (red) expressing cells recorded either in the presence (dotted lines) or absence (solid lines) of extracellular Mg(PDF)Click here for additional data file.Figure S5+/+CNNM4 and −/−CNNM4 mice were stained with toluidine blue. Bar, 100 µm. (B) Retinal sections were stained with the indicated antibodies or lectins. Bars, 100 µm ; 50 µm ; 10 µm . (C) Representative ERG waveforms recorded from 2- and 6-month old +/+CNNM4 and −/−CNNM4 mice. Scotopic and photopic ERGs with stroboscopic stimuli of 1.0 log cd-s/m2 are shown. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; OPL, outer plexiform layer; OS, outer segment; RPE, retinal pigment epithelium.Histological and ERG analysis of the retina. (A) Retinal sections from 2- and 6-month old (PDF)Click here for additional data file.Video S12+-imaging analyses. HEK293 cells expressing CNNM4-FLAG (labeled with asterisks) were loaded with Magnesium Green and then subjected to time-lapse imaging analyses by changing the extracellular solution from phase 1 to phase 4 (see the text for detail).Time-lapse Mg(MOV)Click here for additional data file.


Video communication has been shown to create a sense of proximity between young children and parents. To determine if video affords a stronger sense of proximity and engagement than a traditional telephone, the current experiment employed a Separation and Reunion Paradigm with either a video-link or an audio-link available to the separated dyad. Results revealed that during the separation with a video-link, more children remained content to be physically alone than during the audio-link, children played more and displayed more positive affect. This is the first empirical demonstration that video provides a stronger sense of proximity and enjoyment for young children than audio, suggesting that video is a more appropriate medium to meaningfully connect children to relatives during geographical separation. Since its invention in the late 1800s the telephone has played an important role in communication. Today however, internet infrastructure has enhanced communication options such that people are no longer limited to only talking to each other, but can now simultaneously see each other. Video communication has become increasingly popular as a means of maintaining relationships when actual face-to-face contact is not possible  questionnaire which was based on the AQS Waters, . The oriM = 32.6, SD = 5.36). Nine children were aged 2–2.5 years; nine were aged 2.5–3 years; and seven were aged 3 years. The majority of children (n = 22) participated with their mother rather than their father. An additional five children participated but were excluded from the analysis due to technical problems with the cameras. Participants were recruited through online and print advertisements as well as word-of-mouth referrals.Participants included in the analysis were 25  children aged 25.0–42.5 months , audio , free-play episodes , and interaction during the video , audio , free-play episodes . One of the coders was blind to the experimental hypotheses.Cohen’s Kappa revealed substantial inter-rater reliability for play during the video  and were eliminated from further analysis. Since data violated the assumptions required to perform parametric tests non-parametric tests were used.Preliminary statistical analyses indicated that children’s gender, previous video communication experience, and separation order did not have any effect on the dependent variables  than the audio separation (64%) with a medium effect size. .Initial analysis of the contentment data revealed that a large proportion of participants remained content for the entire 300-s duration. Consequently, analyses were performed on the categorical variable “played showed that children played the most during the free-play episode, followed by video separation, and the least during the audio separation, χ2 = 19.432, p < 0.001. Further planned comparisons using Wilcoxon Signed Ranks tests  = 8.313, p = 0.016. Further planned comparisons using Wilcoxon Signed Ranks tests  = 11.293, p = 0.004. Further planned comparisons using Wilcoxon Signed Ranks tests  = 4.829, p < 0.089. Further planned comparisons using Wilcoxon Signed Ranks tests  when they interacted via a video-link than an audio-link (Walther and Bazarova, We do acknowledge some minor but notable limitations to this research. One limitation is that all our participants were deemed to be securely attached to the participating parent – and that our experimental procedures were designed to elicit and assess the behavioral patterns typical of securely attached children. Thus, alternative experiential designs would need to be employed to assess the use of video communication with children with avoidant, ambivalent, and disorganized attachment styles. It should be noted that, although untested, we have no reason to assume that the advantages of video communication over audio with respect to parental propinquity would differ for children with insecure attachment styles.Another limitation is that we are reporting on laboratory-based findings, and it is possible that behavioral and emotional patterns could differ in the home environment. Nonetheless, we believe that the findings of this research support the theorized value of video communication with young children for maintaining relationships. Similar research should eventually be undertaken examining video communication and audio communication in more ecologically valid scenarios.Conversely, an additional limitation is that our design did not tightly constrain the adult contribution to the communication sessions. For example, parents may have communicated differently when communicating with their child via video than via audio-only. Thus, differences in the parental behavior across the separation sessions may have influenced their children’s behavior. While it would be interesting for future studies to evaluate the effects of parental behavior, our results are arguably more ecologically valid because parents were instructed to behave as they typically would during an audio or video communication session.In summary, our findings demonstrate that compared with audio, video allows for more meaningful communication with young children. In addition to providing a sense of proximity and security more akin to physical presence than an audio-link, children were happier during the video-link than audio-link, which in a practical application would influence their motivation to participate. Given that video provides a stronger sense of proximity and engagement than audio, it follows that video communication is a more beneficial tool for maintaining family relationships with children during times of geographical separation.The experiment described here considers a relatively new technology, and examines its appropriateness for young children compared to telephone communication. It highlights the importance of the visual elements of video communication for the child to obtain a sense of proximity with his or her parent. Given that extended families are increasingly separated by large distances, thus limiting traditional face-to-face contact (De Bruycker, Conceived and designed the experiment: Jordy Kaufman, Joanne Tarasuik, Roslyn Galligan. Performed the experiment: Joanne Tarasuik. Analyzed the data: Joanne Tarasuik, Jordy Kaufman. Wrote the paper: Joanne Tarasuik, Jordy Kaufman, Roslyn Galligan.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.


The use of oral contraceptives was found only in 2 unclassified HCAs (29%). Rather low percentage of female patients and use of oral contraceptives appear to be common clinicopathological features in Japan and also East Asian countries. Furthermore, a group of possible inflammatory HCAs characterized by strong immunoreactivity for serum amyloid A (SAA) was found in patients with alcoholic cirrhosis. The inflammatory HCA/SAA-positive hepatocellular neoplasm in alcoholic cirrhosis may be a new entity of HCA, which may have potential of malignant transformation. Further studies are needed to clarify genetic changes, monoclonality, and pathogenesis of this new type of hepatocellular neoplasm.Hepatocellular adenoma (HCA) is generally a benign hepatocellular tumor arising in a nonfibrotic/cirrhotic liver, and recently four major subgroups were identified based on genotype and phenotype classification from Europe. HCA is rare in Asian countries including Japan, and there have been few studies regarding the subgroups of HCA in Japan. We surveyed subgroups of HCA in 13 patients (7 women) in Japan, based on the phenotypic classification. As results, we identified 2 hepatocyte nuclear factor (HNF) 1 Eighty-or gp130 . In inflor gp130  and addior gp130 . Genotypaterials –6. Less aterials –11. Therβ-catenin, and serum amyloid A (SAA). LFABP-negative, GS-positive and/or nuclear β-catenin-positive, and SAA- positive HCAs are regarded as HNF1α-inactivated, β-catenin-activated, and inflammatory HCAs, respectively [Immunohistochemical markers used for the phenotypic classification include liver fatty acid binding protein (LFABP), glutamine synthetase (GS), ectively –6. The rα-inactivated HCAs (2 women), two β-catenin-activated HCAs (one woman), 5 inflammatory HCAs (one woman), and 4 unclassified HCAs (3 women). The use of OCs was found in only 2 unclassified HCAs (29% of women). β-catenin-activated HCA, 4 inflammatory HCAs (36%), and 2 unclassified HCAs (50%). Six patients (46%) had multiple HCAs; one HNF1α-inactivated HCA, 2 inflammatory HCA, and 3 unclassified HCAs. All multiple HCAs were of the same type in each case. The tumor was significantly larger in β-catenin-activated HCAs than in other subtypes. The association of hepatocellular carcinoma was seen in only one case of unclassified HCA. A definite diagnosis of HCC was made based on cytological abnormalities, a loss of reticulin fiber in a focal area in this HCA. In addition, this area showed the immunoreactivity for glypican-3, which is a marker of HCC.Patients with HCAs included 6 men and 7 women and their age ranged 15–68 yrs (41.0 ± 13.9 yrs). We identified 2 HNF1α-inactivated HCAs is rather low.α-inactivated type. In addition, HCC, not HCA, is generally suspected for hepatic nodules arising in men. In fact, partial hepatectomy was performed in male patients in our survey, since HCC was suspected. Same nodules might be diagnosed in HCA in women and would be followed up without resection. The previous study in Japan reported a higher rate of male patients with HCAs associated with predisposing factors, such as glycogen storage disease and anabolic hormone treatment for aplastic anemia [Compared with previous studies reported from Western countries –5, 13, ic anemia . Such prc anemia . Taken tTumor size ranged 1–31 cm and mean of tumor size was 5.3 cm, 11.3 cm, 8.3 cm, and 7 cm in each study  3, 9–11–119–11. β-catenin mutation [The malignant transformation of HCA to HCC has been described in previous studies , 14–16. mutation , 3, 14. mutation , 14. Themutation .α-inactivated HCA was lower than the study from France [α-inactivated, 18%; HNF1α-inactivated and β-catenin—activated 18%, β-catenin—activated, 12%; inflammatory HCA, 29%; unclassified, 24% [α-inactivated HCA may be a feature of HCAs in Japan. Since HCA associated with mutant HNF1A, a major type of HNF1α-inactivated HCA, occurs almost exclusively in women [We have reported for the first time a proportion of subgroups of HCAs in Japan and Asian countries, to our knowledge . In our m France , 3. Receied, 24% . A lowerin women , higher in women . TherefoOur previous study highlights a group of hepatocellular nodules in alcoholic cirrhosis which conflicts with the general concept of “HCAs” . In factIt is well known that alcohol is a risk factor of HCC. In addition, metabolic syndrome is a newly identified risk factor in chronic liver disease and HCC –23. AlcoClinical and pathological features of HCAs appear to be different in East Asia including Japan and Western countries. Lower percentage of female patients and use of OCs may be common characteristics of HCAs in East Asia including Japan. Our recent studies highlight a characteristic group of hepatocellular neoplasms arising in alcoholic cirrhosis, which share features with inflammatory HCAs. These SAA-positive hepatocellular neoplasms may be a new type of hepatocellular tumors having a potential of malignant transformation in alcoholic patients.


Despite a high number of cases, deaths have decreased 98% since the 1980s. Shigella spp.–related deaths decreased substantially during a period without Shigella spp.–specific interventions. We speculate that nonspecific interventions, e.g., measles vaccination, vitamin A supplementation, and improved nutrition, may have led to the reduced number of shigellosis-related deaths.In 1999, a review of the literature for 1966–1997 suggested that ≈1.1 million persons die annually of shigellosis, including ≈880,000 in Asia. Our recent review of the literature for 1990–2009 indicates that ≈125 million shigellosis cases occur annually in Asia, of which ≈14,000 are fatal. This estimate for illnesses is similar to the earlier estimate, but the number of deaths is 98% lower; that is, the lower estimate of deaths is associated with markedly reduced case-fatality rates rather than fewer cases.  On the basis of studies published during 1966–1997, they estimated ≈1.1 million shigellosis-related deaths annually, resulting from ≈164.7 million cases. Of these, ≈163.2 million cases occurred in developing countries, ≈80% of which occurred in Asia  . To enable comparison over time, we adopted the methods of the previous review on shigellosis . Published rates of diarrhea for each of the 4 strata were used to estimate the diarrheal disease incidence. An increase in the severity of a patient’s illness influences the proportion of diarrheal episodes attributable to Shigella spp. This correlation can be presumed to strengthen as the proportion of Shigella infections increases because sampling progresses from cases of diarrhea detected by household surveillance to those among outpatients to persons admitted to hospital  by the percentage of diarrhea cases from which isolated . For old setting .We adopted the estimates of Kosek et al. . These eShigella spp.–related diarrhea cases for the specified age group. An overall median percentage of shigellosis was then calculated for each stratum and multiplied by the total number of diarrheal cases in the stratum to derive the number of shigellosis cases in each stratum. In addition to the median, a weighted mean with 95% confidence intervals (CIs) was calculated for these analyses by using Freeman-Tukey transformed proportions. Weights used were equal to the inverse standard errors of these transformed proportions . Because only 1 study broke this rate down into the <1-year and 1–4-year ranges, the median of the 2 values for the combined range was calculated. The median frequencies of Shigella spp. isolation rates from persons with diarrhea reporting to the treatment facilities were 5.8% (range 2.4%–9.3%) among children <1 year of age and 9.4% (range 2.4%–23.5%) among children 1–4 years of age. The weighted mean of the combined group was 6.6% (95% CI 6.0%–7.2%). Details of these studies are found in The median frequency of Approximately 39,669,000  shigellosis cases occurred in children <5 years of age in the community and 6,671,000  in treatment facilities, totaling 46,717,000  cases among Asian children <5 years of age annually. The proportions of cases with shigellosis are detailed in Shigella infections among children 5–14 years of age and persons >15 years of age were ≈20,049,000  and ≈58,576,000 , respectively.The median percentage of diarrhea in the community was 4.0% . The median percentages for patients treated at facilities were ≈11.6% (range 4.7%–17.3%) and ≈10.7% (range 4.1%–27%) respectively . .Median CFRs for hospitalized shigellosis patients <1 year and 1–4 years of age and patients >5 years of age were 0.89%, 0.01%, and 0 respectively , accordiShigella infection among children occurred in a health center  cases result in death. Children <5 years of age are at highest risk for Shigella spp.–related deaths may have decreased substantially since the 1980s, even in the absence of specific interventions against shigellosis. We believe the latter explanation best explains the large difference in estimates of deaths.With such a large difference in estimated incidences, one estimate may be more accurate than the other. Alternatively, S. dysenteriae, suggesting that this low CFR may not be limited to Asia  different  reportedly having high sensitivities may detect more infections  also have benefited children with dysentery who may be receiving treatment more quickly than they previously did.The remarkable 98% decrease in deaths from shigellosis in the absence of a Shigella spp., may decrease.The findings from our review may provide lessons regarding other infectious diseases. Approximately 50% of deaths among children <5 years of age have malnutrition as an underlying cause (Shigella spp. are a challenge for vaccine developers, but shigellosis remains high, and increasing resistance to antibiotic drugs continues to make treatment difficult. An effective Shigella spp. vaccine may have substantial benefits, but our study suggests that Shigella spp.–related deaths can be, and have been, substantially reduced with currently available interventions and that such interventions do not need to be Shigella spp. specific.The 4 species and numerous serotypes of Proportion and total number of diarrheal episodes data tables.


Literatures on prevalence and factors associated with malnutrition among peoples living with HIV/AIDS are limited in Ethiopia and not well documented either. The proper implementation of nutritional support and its integration with the routine highly active antiretroviral therapy package demands a clear picture of the magnitude and associated factors of malnutrition. The objective of this study is, therefore, to assess the prevalence and factors associated with malnutrition among peoples living with HIV/AIDS.2 was defined as malnutrition according to World Health Organization (WHO). Binary logistic regression was used to assess association between different risk factors and malnutrition. Confidence interval of 95% was considered to see the precision of the study and the level of significance was taken at α <0.05.Institution based cross sectional study was conducted in Dilla University referral Hospital including adult HIV patients who were in highly active anti retroviral therapy. Interview administered questionnaires were used to collect data on socio demographic factors. Besides, HIV related clinical information was extracted from anti retro viral therapy data base and clinical charts. The nutritional status of the patients was determined by Body Mass Index (BMI) where BMI < 18kg/mA total of 520 patients were included in the analysis. The overall prevalence of malnutrition was 12.3% (95% CI 9.5–15.0). After full control of all variables; unemployment , WHO clinical stage four , gastrointestinal symptoms  and previous (one) opportunistic infection , and two & above previous opportunistic infections  were significantly associated with malnutrition. However, moderately poor economic condition was found to be protective factor for malnutrition .Unemployment, WHO clinical AIDS stage four, one & more number of previous opportunistic infections and gastrointestinal symptoms were found to be important risk factors for malnutrition among People Living with HIV/AIDS. From this study it has been learnt that nutritional programs should be an integral part of HIV/AIDS continuum of care. Furthermore, it needs to improve household income of PLHIV with employment opportunity and to engage them in income generating activities as well. Both Human Immunodeficiency Virus (HIV) and malnutrition can independently cause progressive damage to the immune system. The former increases susceptibility to infection, morbidity and mortality through opportunistic infections, fever, diarrhea, loss of appetite, nutrient malabsorption, and weight loss -3. FurthThe advent of a generalized HIV/AIDS epidemic in combination with drought and food crises exacerbated the famine across many parts of Africa . HIV/AIDEmpirical evidences on malnutrition among People Living with HIV (PLHIV) have shown that socio demographic factors such as gender, employment, income, drinking water and sanitation were closely related determinants of nutritional status. Additionally, gastrointestinal complications, number of previous opportunistic infections and World Health Organization (WHO) clinical AIDS stage were reported to be risk factors for malnutrition among PLHIV -13.HIV/AIDS and malnutrition combine to emasculate the immunity of many Ethiopians . This haHigh rates of malnutrition in Ethiopia worsen the impact of HIV and pose significant challenges to HIV care and treatment programs . A nutriTaking this complex nature of the problem in to account this study was carried out to assess the prevalence of malnutrition and associated factors in PLWHA. It is our strong belief that the results of this study provide valuable information to strengthen HIV/AIDS continuum of care.3 or if they fulfill WHO clinical AIDS stage III or IV.Dilla University referral Hospital is found in Dilla City administration which is located 360 kilometer far away from the capital city, Addis Ababa, in the south of Ethiopia. It is the public hospital which is an affiliate of Dilla University providing training for health sciences student in a range of disciplines. Additionally, the hospital provides higher level of clinical care for nearly a million of catchment area populations. Since 2005, the Hospital has been providing highly active antiretroviral therapy (HAART) for PLWHAs. During the study period , about a total of 3312 subjects had been enrolled in chronic HIV/AIDS care and 1585 patients were on HAART. According to the national guideline, ART shall be initiated for eligible patient. The eligibility of the patients is determined either if their CD4 cell count is < 200/mmCross-sectional study design was conducted including 520 PLWHAs who were 18 years or above. The sources population was a group PLWHAs who had been enrolled in Dilla University Referral Hospital in HAART. The study subjects were drawn by systematic sampling technique from ART registration data base from April 2011 to March 2012.3 severe, 200–499 cells/mm3 as moderate and >500 cells/mm3 as mild. Patients’ medical chart was reviewed for extraction of AIDS’ clinical stage data and history of previous opportunistic infections (OIs) in the last 6 months. In addition, adherence to HAART was extracted from the medical chart of individual patients which was registered during their monthly spell of follow up. Similar to the previous opportunistic infection, adherence status is delimited to the last six months follow up time. However, self reported adherence measurement technique has been used by asking the patients about the number of times they have missed taking their pills each month and recorded. In this study, the mean adherence to HAART for each eligible record was operationally defined as “good adherent” if the average adherence was greater than 95% and “less-adherent” if it was ≤ 95%.Socio-demographic details such as age, gender, residence, employment status, level of education, marital status were collected using interview administered questionnaire. Similar instrument was used for the collection of gastrointestinal symptoms’ data and side effect of ARVs in the past six months from each patient. Poverty status was assessed by index of socio economic status which was measured by summation of items of possession. In this study, it was measured by giving a score of “1” for possessing each of 22 items in the list . The sumThe data collected from the respondents was entered in to Epi info version 7 and imported to SPSS for windows-version 16. The data analysis ranged from the basic description of outcomes to the identification of statistically significant associations. First, the basic descriptive summaries of patients’ characteristics and outcome of interest was computed. Accordingly, simple frequencies, measure of central tendencies and measure of dispersions were scrutinized. Second, bivariate analysis and multiple logistic models used to show the relation between malnutrition and various associated factors. Finally, all explanatory variables that were significantly associated with the outcome variable in the bivariate analyses (P < 0.05) were entered in to stepwise logistic regression model to identify independent predictor of malnutrition. Confidence interval of 95% was used to see the precision of the study and the level of significance was taken at α <0.05.2 were examined by an ART clinician and they had been given a dietary counseling to address areas of their specific concern as it was revealed by the screening tool.The study protocol was reviewed and approved by Dilla University ethical clearance committee. Before data collection, an informed consent was obtained from respondents. Privacy and confidentiality were also maintained throughout the data collection, analysis, and manuscript preparation. Subjects with BMI < 18.5kg/mA total of 520 patients were participated in the study. Majority of the patients were in the age group 30–39 years, with mean age of 33.9 SD = ±8.13) years, and the majority (59%) were women. The greater part of the respondents were currently married (57.7%). About three forth (73.3%) of the study population were employed  for male and 17 (SD = ±2.84) for female. Over all prevalence of malnutrition was 12.3% (95% CI 9.5–15.0). While 7.0% (95% C I 3.60–10.40) of the males had malnutrition, the proportion among females was 16.0% (95% CI 12.89–20.10). The prevalence of malnutrition was increased when the age of the study subjects was increased. For instance, in the age group of less than 30 year-olds, the prevalence was 9.0% (95% CI 4.65–13.35); among the 30–39 year-old it was 13.3% (95% CI 8.87 –17.73); amid 40–49 years old it was 14.3% (95% CI 7.47–21.23); and in the age group of 50 years of age and older it was 16.7% (95% CI 3.35–30.05).2. In the same way, those patients who had manifested gastrointestinal symptoms and those patients who had poor adherence to HAART has shown a BMI score less than 17kg/m2.Table Tables With regard to marital status, a greater number of malnourished subjects were found in the group of the widowed (19.1%) followed by the married ones (11.3%). Being a widow  was significantly associated with malnutrition, but this association was not maintained after adjusting for all independent variables .Concerning literacy variables, it has been found that only illiterate educational status were a risk factor for malnutrition, . Nonetheless, after controlling for all other important variables the association was no longer significant . With reference to employment status, the proportion of malnutrition was higher (23.7%) in unemployed group compared to those employed (8.1%), this difference was statistically significant as well  for unemployed as compared to their counterpart. Moreover, the association remained statistically significant after controlling for other variables . In the same way, moderately poor economic status was found to be protective factor of malnutrition .WHO clinical stage four was found to have a statistically significant association with malnutrition . Independent of all other variables, the result was remained an important risk factor for malnutrition . Those who had poor adherence to HAART in the past six month had a higher risk of developing malnutrition and was statistically significant  although controlling for all other independent variables nullified the association .In spite of the fact that the proportion of malnutrition was higher; (20.5%) among those with severe CD4 cell count and (12.8%) among those in the mild and (9.9%) among those in moderate CD4 cell count category, the association was not statistically significant. The bivariate analysis has revealed the crude odds ratios of  and  for severe and moderate CD4 cell count, respectively.Number of previous opportunistic infections (OIs) showed a significant association with malnutrition after fully adjusting it for all variables. Being having one diagnosis of previous OI had a higher risk for developing malnutrition  and having two or more diagnoses of OIs further increases the likelihood  of malnutrition as compared to those with no previous diagnosis of OIs in the past 6 month. Likewise, independent of all other variables gastrointestinal symptoms (GIS) had significant association with malnutrition. Those patients with one or more GIS had a higher risk of developing malnutrition  as compared to those with no GIS.Meta-analysis from 11 sub-Saharan African countries indicated that the prevalence of malnutrition in Ethiopia among HIV-infected women was 13.2% . It is a2 for male which is almost similar with the general Ethiopian male population of mean BMI of 19kg/m2, but the mean BMI of 17 for female in this study is lower than the general Ethiopian women population of mean BMI 20 [The mean BMI was 19.5kg/mThe higher risk of developing malnutrition in unemployed subjects found in this study is agreed with other study  where unIndependent of all other variables, WHO clinical stage four has significant effect on the likelihood of malnutrition development. Malnutrition is usually encountered at the advanced phase or end of the HIV infection course . An anthConsistent with other findings ,25,26 thThis study did not assess the effect of each opportunistic infection on the nutritional status of the study subjects. Nevertheless, it has been learnt that the number of previous opportunistic infections were independent risk factors of malnutrition. This finding is well supplemented by similar other studies conducted by international agencies and researchers ,9,11. HIOne of the strengths of this study was the use of large sample size and assessment of some important clinical factors associated with malnutrition. To avoid recall biases, medical charts and ART data base were triangulated with the primary data collected by structured interview administered questionnaire. However, the cross-sectional nature of the study limits the investigation to the level of the association between determinants and outcomes of interest . Hence, it’s impossible to get information about causal relationship in the majority of associated factors. Likewise, further comparative study, between HIV-infected and non-HIV infected persons that could explore more risk factors for malnutrition is recommended.The results of this study provide data on the characteristics of nutritional status of HIV-positive patients and important associated factors. To mention few, employment status, poverty status, WHO clinical staging, number of previous opportunistic infections and gastrointestinal symptoms were among the imperatives. It has been learnt that malnutrition & its problems in HIV patients are complex & interwoven; no single recipe exists as solution either. Hence, there is a prompt need to integrate nutritional care in comprehensive continuum of HIV care. As well, it’s needless to argue about improving household income through creating employment opportunities and engaging the needy unfortunates in amenable income generating activities could possibly alleviate these predicaments.The authors declare that they have no competing interests.SH contributed in the generation of the topic, preparation of proposal, data collection, analyses and development of the manuscript. GT contributed in reviewing the proposal, assisted in data collection, analyses and critical review of final manuscript. HT contributed in critically reviewing the proposal, the manuscript and processed publication. All authors read and approved the final manuscript.


This mixed-methods study qualitatively (n = 13—convenience) explored contextual factors influencing decisions to drink responsibly, and quantitatively (n = 729—random) assessed the prevalence of these factors and whether they varied as a function of sex and binge-drinking status. Data were collected in 2007 among Texas college students. The constant comparison model was used to analyze the qualitative data, while one-way ANOVAs and logistic regression were employed to assess the quantitative data. Effect sizes are reported for all significant ANOVA interactions. Psychometric properties are supplied for all quantitative scales. Implication and limitations are noted, and future research directions discussed. Even though 4 out of 10 U.S. college students engage in heavy episodic alcohol consumption , only a after college, it is imperative that college students avoid succumbing to the harmful alcohol-related consequences associated with their drinking patterns during college. In fact, college students themselves recognize the need for such educational/interventional programming aimed at reducing negative alcohol-related consequences. For instance, after conducting a qualitative investigation into the methods college students employ to minimize alcohol consumption-related harm to both themselves and others, drink responsibly [emphasis added]…” (p. 252).While it is possible for students to transition out of their excessive drinking behaviors While researchers have attempted to include responsible drinking as a behavioral outcome in their interventions, so far these attempts have suffered from serious methodological limitations. Specifically, researchers are “consistently inconsistent” in their efforts to identify explicit characteristics of responsible drinking , p. 301.Although an initial investigation into the Specific beliefs and behaviors college students associate with responsible drinking has been conducted , to dateAs a caveat, we wish to point out that this study does not address “moderate drinking” , a constThis study employed a partially mixed sequential dominant status design , or a miThe initial phase of this investigation sought to qualitatively explore the contextual factors infuencing one's responsible drinking practices. Due to the dearth of systematic, published investigations into responsible drinking, this phase encompassed a series of less structured focus group sessions. Less structured groups are an ideal choice when researchers do not have prior knowledge/insight into the topic they are investigating . An “emeParticipants. Participants were recruited from several health promotion core-content courses offered at a large, four-year public university in Texas. The first author visited randomly chosen class sessions to provide information regarding the purpose and overall objectives of the study. Once informed about the objectives, students could indicate their interest in participating in the study, by providing (on an index card) their name, e-mail address, and the most convenient day(s) and time(s) for meeting with a focus group. The final sample size comprised 13 individuals, two men and 11 women. The majority of the sample was Caucasian (n = 11), with nominal representation of African-Americans , and Hispanics .Data Collection. Prior to beginning each focus group session, participants reviewed and signed an IRB-approved informed consent form. As outlined in the consent document, each session was audio-taped. In total, four focus group sessions and three individual interviews (necessary in order to accommodate participants' scheduling conflicts) were conducted. The first author facilitated each focus group session and conducted each interview. During focus group or interview sessions, participants were asked to discuss the contextual factors infuencing their responsible drinking behaviors. Specifically, participants were asked a series of questions including, but not limited to, “Can you think of any barriers and/or obstacles that would prevent you or your peers from drinking re-sponsibly?”, “What types of situations would impact your ability to drink responsibly?”, “Can you think of a situation in which it would be impossible for you or someone else to drink responsibly?”, and “Can you provide some examples of how you could drink in a more responsible manner?” During discussions, participants were probed to provide Specific norms, attitudes, and/or beliefs associated with their contributions. All identifying characteristics or personal descriptions  were removed from the typed transcripts as well as from any presented or published accounts of the sessions, to ensure confidentiality.Data Analysis. At the conclusion of each session, typed transcripts were developed from the audio-recordings. Using the constant comparison model ((1)FINDINGS: Contextual Factors Infuencing Responsible Drinking. Overall, the emerging contextual factors infuencing the practice of responsible drinking were either intrapersonal  or environmental  in nature. Grounded in the Ecological Perspective Intrapersonal Factors: Personal Responsibilities. Participants believed their school-related obligations represented a salient factor impacting their responsible drinking practices. For example, the following days' school schedule emerged as a factor affecting decision-making: “Do I have class at eight in the morning? If so, maybe I shouldn't stay out until four O'clock.” Another participant echoed the impact of school-related factors, saying:You may go somewhere  knowing you have a test the next day and say “I am just going to go for a little bit. I will just have one or two drinks.” But then if you have one or two drinks, you might come home and be tired, not study for that test like you were supposed to, and do poorly.I know that if I go somewhere and I drink and then I have to come home and take care of my kids I know I cannot do it well if I have had drinks, or maybe the next morning I will not be able to be as attentive as I would have been if I didn't have drinks.In addition to school-related factors, scenarios involving personal accountability and responsibility also emerged. One such example was the health of an unborn baby, or “not drinking during pregnancy.” This participant elaborated on the manner in which her motherly duties influenced her drinking behaviors, stating:on model  to sort on model .(1)FIND(1)Emotional Status. An individual's emotional state/status also emerged as a factor potentially impacting responsible drinking behavior. This subtheme revolved around feelings, such as anxiety, stress, or depression. For example, one participant stated, “sometimes you are feeling depressed or stressed out and you want to get away from that, and that causes people to drink more than they usually would.” Participants cited Specific, hypothetical, instances that could impact one's decision to drink responsibly. Some of these instances inhibiting ability and/or decision to drink in a responsible manner, ranged from work-related circumstances [“a bad day at work”] to relationship problems [“boyfriend or girlfriend broke up with them”].(2)Environmental Factors: Monetary Considerations. Participants felt carrying an excess of cash while in a social setting, such as a bar, could influence drinking behavior. Specifically, excess money allowed additional drinks, should he/she feel the urge, to be purchased at whim. As a preventative measure, and strategy to limit consumption, participants allocated a set amount of money to an evening's-worth of alcohol. Participants felt this strategy prevented them from giving in to temptation and assisted in limiting alcohol consumption. In other words, persons used money as a meter for determining how much had been (and could be) consumed; once the money was gone, drinking stopped. For instance, one participant revealed “I will only bring enough money for a certain amount of drinks. If you cannot afford to have more, you aren't tempted to drink more because you don't have the money for it.” Another participant echoed this sentiment, stating “I make sure not to bring a bunch of money to the bar that I could spend on drinks.” In addition to applying this concept in a social setting, others implemented this strategy when purchasing alcohol for consumption at home: “I only like to buy a certain amount because I will know exactly how much I am going to drink. That makes me feel very comfortable.” When probed to develop this point, the participant offered the following analogy: “It is like cooking extra; if you don't cook extra food then you will not eat more.” Similarly to cash, the utilization of credit cards emerged as an influence on responsible drinking. Specifically, the “open tabs” that credit cards allow was characterized as a detriment to responsible drinking behavior. By keeping an “open tab,” participants felt it was easy to lose track of both the amount spent on alcohol, as well as the number of drinks consumed. For these reasons, one participant identifed credit card use at a bar as “terrible.” This individual elaborated, stating “my friends get open tabs with their credit cards and by the end of the night they are like ‘wow, I did not realize this!”’(3)Surrounding Environment. Another external factor potentially impacting responsible drinking included the surrounding environment. This notion applied not only to the location where drinking occurs , but also to the individuals present at the time . With regard to the physical environment, participants felt one needs to “be in a safe setting.” Participants depicted a safe environment as, “go to someone's house to drink all night and stay the entire night there.” In addition to considering where one will drink, participants insisted the individuals present should also be considered. As one person stated, “If you are going to drink alcohol you should feel comfortable and trust people you are around.” Thus, participants felt it was important to consider not only where one will be drinking, but also who is present and how well they're known. One participant asserted,You need to make sure that you are around people you are familiar with. You have to trust the people you are around. If you are just in a bar getting drunk, you do not know the people you come into contact with.Elaborating on the importance of one's drinking environment, and those in it, multiple female participants highlighted the dangers of leaving a drink unattended: “Watch your drink. Do not set them down because people could always put something in them.” Multiple female participants were wary of other individuals and fearful of being dosed with a “date rape drug.” These admissions clearly point to a gender-related concern, as none of the males in the current sample discussed or alluded to consequences associated with leaving a drink unattended.(4)Drinking Games. The current sample also identifed participation in drinking games as precluding drinking in a responsible manner. (5)Other People. Lastly, other persons were also cited as impacting one's decision to drink responsibly. One form of influence alluded to in our sample was peer pressure. Specifically, participants provided insight into two distinct types of peer pressure infuencing responsible drinking: indirect and direct. Thus, personal responsibilities, whether school or family-related, emerged as an intrapersonal factor infuencing one's decision to drink responsibly.Instances exemplifying direct peer pressure among our sample were primarily related to hazing. However, the apparent pressure applied on participants was not restricted to brute force. For instance, the simple gesture of offering a drink could have profound influence: “I think a single girl just going to the bar with her friends to have fun and plans on not drinking, then a cute guy buys her a drink, I think that she would probably take it.” Thus, whether being propositioned or forced to consume alcohol, one's ability to drink responsibly was cited as being hindered by direct peer pressure. Examples of indirect peer pressure primarily stemmed from social norms and group membership. One participant explained, “if all your friends' activities revolve around drinking, then it becomes part of hanging out. You don't want to miss out on time with your friends so you drink too.” Said differently, “If everyone is at the party and everyone is drinking then you might feel left out.”A subtheme associated with the influence others exert on responsible drinking behavior emerged in the concept of a “designated caretaker.” Participants in our study defined this drinking “buddy-system,” as having another individual, whom the drinker trusts, make decisions for the drinker while he/she is intoxicated. This practice was identifed as a method not only to ensure the drinker's safety, but also to accomplish responsible drinking. As one participant explained, a caretaker is “there to watch out for you and make sure that you are going to have a safe way to get home and that no one can take advantage of you.” At its core, this theme centered on the concept of having a known, trusted friend who could “make wise choices about any situation that may arise that night.” A participant-provided analogy equated a designated caretaker to “a sort of mother hen.”At the conclusion of Phase One, the aforementioned themes were used as the foundation for the development of two quantitative scales. In other words, based upon the participant-provided contributions, a set of items was developed to address the contextual themes emerging from Phase One. Once developed, these scales allowed for the second phase of this study to be completed. Specifically, we (a) assessed the prevalence of the contextual factors among a sample of college students attending a large, Texas, public institution, and (b) determined whether the prevalence of these factors varied as a function of both sex and alcohol consumption.Sample. A randomly selected sample of currently enrolled students was solicited via e-mail to participate. Of the 5,000 invitations sent, 4,985 were received. Of those successfully contacted, a total of 729 (15% response rate) students returned usable surveys. On average, respondents were 22 years of age (SD = 5.49). Females (55%) represented a slight majority in this sample. Ethnically, the majority of the sample was Caucasian (76%); while the remainder were Hispanics (10%), Asian or Pacifc Islander (7%), African-American (2%), Eastern Indian (1%), American Indian or Alaskan Native (0.5%), Middle Eastern (.5%), or Other (2.3%). That said, it is important to note that the demographic (gender and ethnic) distribution of this sample is comparable to the population from which it was drawn. While primarily Caucasian (73%), the university's three largest ethnic groups are also Hispanics (11%), Asians (4%), and African-Americans (3%). Females represent 47% of the institution's student body and were, therefore, slightly over-represented in our sample. Considering the low proportion of individuals in ethnic backgrounds other than Caucasian and Hispanic, for data analysis purposes all non-Caucasian and non-Hispanic ethnicities were collapsed into an “Other” category in order to ensure enough participants were included in each ethnic group.Measures. To assess the prevalence of contextual factors infuencing college students' responsible drinking practices, two distinct scales were employed. All included scale items were developed based upon the themes emerging from the qualitative data from Phase One. For example, on the responsible drinking motivations scale (21 items), respondents were asked to indicate if work- or school-related obligations, monetary concerns, or the actions of others would motivate drinking responsibly. Additionally, as part of the responsible drinking barriers scale (16 items), respondents indicated if being challenged to a drinking contest, peer pressure (both direct and indirect), or performing badly on school assignments would be a barrier to drinking responsibly. For each item in the scales utilized, respondents were asked to indicate whether the given conditions/situations served as a potential motivator/barrier to drinking responsibly (1) never, (2) seldom, (3) some of the time, (4) most of the time, or (5) always. See ns scale 1 items, Before analyzing the data produced from these scales, the internal consistency  of the motivation and barriers scales employed in this investigation were evaluated. With this study's sample, the motivation scale exhibited a Cronbach coeffcient alpha of 0.87 and the barriers scale, a 0.91. Scales exhibiting coeffcient alphas within this range have been deemed “very good” . Both scData Collection. In order to obtain a representative sample, the investigators obtained a list of all currently enrolled undergraduate and graduate students attending the institution. This list contained students' full names and their corresponding e-mail addresses. Using the list as a sampling frame, a random sample was invited to participate in the study via e-mail. Contacted individuals had approximately seven days to complete the online survey before the link would become inactive. Reminders were sent to those who had not yet accessed or completed the survey on days three and five. In order to access the survey, individuals clicked on a hyperlink embedded within the invitation and/or reminder e-mail. Persons clinking on the weblink were subsequently directed to an Information Sheet outlining informed consent matters. A “Go to Survey” link was located at the bottom of the information sheet. By clicking on the “Go to Survey” link, participants were confrming their understanding and voluntary acceptance of the study procedures.Data Analysis. Prior to data analysis, participant non-response was examined. After taking into account non-response due to embedded skip patterns , all motivation scale items exhibited less than 1% missing data and all barriers scale items exhibited no more than 5%. Thus, the quantity of missing data for the measures was quite low. With regard to the pattern of missing data, nonresponse on both the motivation and barrier scales were examined. Independent sample t-tests confrm that respondents with fully completed motivation scales did not significantly differ from those with missing responses with regard to gender  or binge drinking status . Similarly, those who fully completed all the barrier scale items did not significantly differ from respondents with missing responses based on gender  or binge drinking status . Due to the lack of large amounts of missing data, as well as the manner in which data were missing, it could not be justifed to employ complex analytical technique(s) to calculate imputable scores. Moreover, it was determined that data were missing at random (MAR). Researchers categorize data MAR as “ignorable”  (version 18.0), basic descriptive statistics (Mean ± SD) were generated for each item on the two included scales. Sex- and consumption-based differences for each scale were calculated using one-way ANOVA. Cohen's Results. In establishing the extent to which a factor served to facilitate or impede responsible drinking practices, there was a clear distinction between male and female responses across the motivations and barriers scales. Females were more likely to indicate that an item on the motivations scale facilitated responsible drinking, while less likely to indicate that an item on the barriers scale impeded responsible drinking. In other words, males identifed more factors as obstructing responsible drinking practices, while at the same time identifying fewer factors aiding drinking responsibly.F = 4.546, p < .033]; “because I do not want to do anything out of my character I may later regret” ; “because I do not want someone to take advantage of me” ; “because I want to have control over my actions” ; “because I do not want to get nauseous or vomit” ; “because I want to be aware of and understand what is going on around me” ; “because I want to remember what happens” ; “because I want to keep my blood alcohol concentration (BAC) under 0.08%” ; and “because I am with people I do not know very well or in a new environment” . See Women were significantly more likely to identify the following factors as a motivator to responsible drinking: When I drink responsibly, one of my motivations is… “because of my religious convictions” , “I was playing a drinking game” , “I had someone challenge me to a drinking contest” , and “I felt pressured by friends to drink” . See d) are reported in conjunction with all significant group differences presented in both Regarding factors serving as barriers to responsible drinking, women were significantly less likely to identify the following factors as barriers to responsible drinking: The next time I drink alcohol, I would not be able to drink responsibly if… “an attractive person wanted to buy me a drink(s)” ,While previous investigations have documented that males believe responsible drinking behaviors must occur with significantly less frequency when compared to their female counterparts , the quaThere are important limitations that must be considered in unison with the contributions of this investigation. Specific to the qualitative phase, the lack of gender/ethnic diversity among participants and the small sample size are of particular concern. While transcripts indicate saturation was reached [later focus group sessions support early concepts/ideas and do not provide unique contributions ], a moreThe most prominent limitation from the quantitative phase was the low survey response rate. Such a low response rate raises questions of the sample's representation. While the demographic distribution of the sample parallels the population from which it was drawn (with regard to both gender and ethnicity), this does not ensure the absence of selection biases. Nevertheless, low response rates with online, electronic surveys of young adults and professionals, has not been uncommon .Overall, this research builds upon and enhances previous investigations outlining the behavioral beliefs college students have about responsible drinking . To dateCollegiate binge drinking rates are practically the only substance use/misuse rate among young adults that has remained stagnant for nearly the entire past two decades . While nConsidering the scarcity of systematic investigations into responsible drinking practices, or the factors behind one's practice of these behaviors, this initial study opens several avenues for future research. Future inquiries should seek to (a) further flush out how people conceptualize and practice responsible drinking, (b) establish the generalizability of these findings across different academic institutions and geographic regions, and (c) establish additional factors that may serve as a motivator or barrier to responsible drinking. By continuing to examine the contextual factors that influence one's responsible drinking practices, researchers will move one step closer to developing evidence-based strategies designed to promote responsible drinking and diminish the negative health effects associated with excessive alcohol consumption.


Paenibacillus polymyxa CR1 (accession no. CP006941), which consists of one circular chromosome of 6,024,666 bp with 5,283 coding sequences (CDS), 87 tRNAs, and 12 rRNA operons. Data presented will allow for further insights into the mechanisms underpinning agriculturally and industrially relevant processes.Here we report the complete genome sequence of the bacterium  Paenibacillus polymyxa is a species of Gram-positive, sporulating, facultative anaerobes that are widely distributed and have been isolated from various environmental samples , P. polymyxa SC2 (NC_014622), and P. polymyxa M1 (NC_017542) . Here we report the whole genome sequence in order to better elucidate the agriculturally and industrially relevant metabolic processes of P. polymyxa CR1. samples –3. Recenmedicine –10. So f_017542) –13. OrigP. polymyxa CR1 was isolated from culture growth for 24 h at 37°C in nutrient broth using a Sigma-Aldrich GenElute bacterial genomic DNA kit (product no. NA2120). Two libraries with an approximate insert size of 400 bp were prepared for sequencing using the NexteraXT DNA sample preparation kit for the small insert library and the Nextera mate pair sample preparation kit for the pair read library. The resulting sample libraries were purified and their quality evaluated by quantitative PCR (qPCR) and KAPA library quantification kit. These libraries were used as the templates for sequencing using a limited-cycle PCR using Nextera primers.Genomic DNA of de novo using ABySS, Velvet, and SOAPdenovo  are present in the P. polymyxa CR1 genome, as well as genes responsible for plant hormone (indole-3-acetic acid) synthesis, biomass degradation, antimicrobial production, and butanol production . Further analysis and study are required to establish the intricacies of these complicated metabolic and signaling pathways (Genes responsible for nitrogen fixation (oduction , 21, 22.pathways .P. polymyxa CR1 genome will lay a solid foundation for understanding the synthetic pathways and underlying mechanisms. This knowledge will guide genetic modification of metabolic pathways to fully exploit the potential of P. polymyxa CR1 in agricultural and industrial applications.In-depth analysis of the P. polymyxa CR1 has been deposited in NCBI’s GenBank with the accession number CP006941.The sequence of