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Background: Anchovy (Stolephorus sp.) is a commonly used food ingredient due to its high calcium content, which supports craniofacial growth. Calcium stimulates the formation of osteoblasts, which produce osteoprotegerin (OPG). OPG binds to RANKL, blocking RANKL-RANK bonding and limiting osteoclast development., Objective: The objective of this study was to analyze OPG expression in mandibular bones to assess the potential calcium content of anchovies., Methods: Three groups of 27 male Wistar rats were created: control, anchovy, and milk. After 40 days, the rats were decapitated, and their mandibular bones were surgically extracted, decalcified, and prepared for microscopic examination. The results showed a significant difference in OPG expression of rat mandibles between control, anchovy, and milk groups ( P < 0.05), as determined by one-way analysis of variance (ANOVA). Tukey's HSD test revealed a significant difference in the average quantity of rat mandibular OPG expression between the control group and each of the anchovy and milk groups, with probability values of 0.00 and 0.003 ( P < 0.05), respectively. However, the average level of OPG expression in anchovy and milk groups did not differ significantly, as indicated by the probability value of 0.064 ( P > 0.05)., Conclusion: The mandibular development increases after feeding anchovies compared to the control group. Anchovy is not statistically superior to milk in terms of increasing mandibular development. However, as a high-calcium food, anchovy is well-suited to support children's craniofacial development and growth., Competing Interests: There are no conflicts of interest., (Copyright: © 2024 Journal of Oral and Maxillofacial Pathology.)

Smoking is related to vascular aging. However, the hazardous effect of e-cigarette is often debatable, with limited studies available. In contrast, moderate-intensity aerobic exercise is well known to decrease aortic stiffness. We provide novel research to determine the effect of e-cigarette and aerobic moderate-intensity exercise on the aortic structure of Wistar rats. A total of 26 male Wistar rats (Rattus norvegicus) 8 weeks aged, 200-250 g b.w., were randomly divided into 4 groups, namely, K0 (normal rats), K1 (rats were given moderate-intensity aerobic exercise by animal treadmill 20 m/30 min), K2 (rats were given e-cigarette with 6 mg nicotine, 40% propylene glycol, and 60% vegetable glycerine 30 min for 5 days/week), and K3 (rats were given e-cigarette and moderate-intensity aerobic exercise). After exposure for 6 weeks, all animals were sacrificed to isolate the aorta for histopathological analysis with hematoxylin-eosin stain to evaluate the elastic fiber layer and intimal-medial thickness. The Verhoeff-Van Gieson staining was done for quantification elastic lamina fragmentation. Our study found that the e-cigarette group had the highest elastic lamina fragmentation among groups (8.14 ± 2.85). The exercise only group showed the lowest elastic lamina fragmentation (2.50 ± 1.87). Fragmentation in the e-cigarette and exercise group was higher than in the exercise only group (5.83 ± 0.753 vs. 2.50 ± 1.87, p = 0.002). There is a significant difference of NO serum between four groups. The result of post hoc analysis using LSD showed that there is a significant difference of NO serum between K0 and K2, K0 and K3, K1 and K2, and K1 and K3. Therefore, our research demonstrated that the most injury of aorta elastic lamina was in the group that was exposed to e-cigarette that leads to vascular aging while exercise is not yet proven to reverse this effect., Competing Interests: There is no conflict of interest in this study., (Copyright © 2023 Vito A. Damay et al.)

Stocking broilers at a high density has been a strategy to optimize the area of the cage and hence increase the efficiency of broiler production. If the environmental (microclimate) conditions and rearing management are not properly managed, stocking broilers at a high density may, however, result in stressful conditions that are harmful for the production, health and welfare of broilers. To ameliorate these unfavorable effects of overcrowding stress, dietary interventions have been conducted. Probiotics, prebiotics, synbiotics, plant-derived products, vitamins, propolis, amino acids, fatty acids, etc. have been supplemented in diets to deal with the harmful impact of stress induced by a high stocking density of broilers. This review covers the detrimental effects of overcrowding-induced stress on broiler development and attempts to ameliorate those negative effects by dietary interventions., Competing Interests: The contact author has declared that there are no competing interests., (Copyright: © 2022 Sugiharto Sugiharto.)

The study investigated how sprouted-papaya seed meal (SPSM) a total of affected the growth and carcase traits of broilers. Based on a completely randomized design, 390 day-old Lohmann broiler chicks were allotted to CONT (chicks provided with control diet), SEED25 (diet containing 2.5% papaya seed meal), GERM1 (diet containing 1% SPSM), GERM25 (diet containing 2.5% SPSM) and GERM5 (diet containing 5% SPSM). Body weight and feed intake of chickens were weekly recorded from 14 to 36 days of age, while birds (six birds per treatment group; 30 birds in total) were slaughtered at day 36. Feeding SPSM up to 5% did not impair ( p  > 0.05) broilers' growth or feed intake. At 5%, SPSM compromised ( p  < 0.05) feed conversion ratio (FCR) and reduced breast meat proportion. SPSM at 2.5% in diets had no ( p  > 0.05) detrimental effects, while papaya seed meal at the same proportion lowered ( p  < 0.05) final body weight, weight gain, and cumulative feed intake. Overall, SPSM may be incorporated in broiler rations up to 2.5% with no harmful effects on growth, feed intake, FCR and carcase traits of broiler chickens., Competing Interests: No potential conflict of interest was reported by the author(s)., (© 2021 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.)

The study investigated the effect of acidified turmeric, black pepper or its combination on growth and meat quality of broilers. The Averrhoa bilimbi Linn. fruit filtrate was used to acidify the herbs. A number of 392 day-old Lohmann broiler chicks were randomly distributed to four groups, including CONT (control diet), TRMC (diet supplemented with 1% acidified turmeric), BLPR (1% acidified black pepper) and TRPR (1% acidified turmeric and 1% acidified black pepper). Body weight, feed intake and feed conversion ratio (FCR) were weekly recorded. Internal organ weight and carcase traits were determined at day 35. The CONT and TRMC showed greater (p < 0.05) weight gain than BLPR and TRPR. The FCR was lower (p < 0.05) in TRMC than in BLPR and TRPR, but did not differ from CONT. The gizzard was greater (p < 0.05) in BLPR than that in CONT and TRMC. The BLPR had smaller (p < 0.05) pancreas than other chickens. Abdominal fat was lower (p < 0.05) in TRMC, BLPR and TRPR than that in CONT, of which BLPR was the lowest. Drumstick was greater (p < 0.05) in BLPR than in CONT. CONT had lighter and less yellow (p < 0.05) breast meats than other broilers. In thigh meats, the lightness (L*) values were higher (p < 0.05) in CONT than in TRMC and BLPR. The yellowness (b*) were lower (p < 0.05) in CONT than in TRPR meats. In conclusion, acidified turmeric reduced abdominal fat deposition and improved meat quality of broilers., Competing Interests: No potential conflict of interest was reported by the authors., (© 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.)

Background: Diabetes self-care reminders are widely applied to improve self-care among diabetes patients. Unfortunately, there is a lack of self-care reminders for diabetes patients who live in rural areas of developing countries with limited resources., Aims: The study evaluated the feasibility of the Diabetes Self-Care Calendar as a reminder tool for self-care., Methods: The study was conducted in a community health centre in Pekalongan Regency, Indonesia. The Diabetes Self-Care Calendar was developed as a reminder tool following the American Association of Diabetes Educator self-care guidelines. The feasibility of the calendar was evaluated using the quality assessment questionnaire that assessed accessibility, acceptability, effectiveness, efficiency and appropriateness. The participants' qualitative feedback was organised into themes., Results: A total of 10 patients with type 2 diabetes mellitus participated in the study. The quality of the calendar was evaluated in terms of accessibility (m = 9.5; range = 0 - 10), acceptability (m = 9.7; range = 0 - 10), effectiveness (m = 9.3; range = 0 - 10), efficiency (m = 9; range = 0 - 10) and appropriateness (m = 9; range = 0 - 10). The feasibility of the calendar was m = 9.3 (range = 9 to 9.7). Three themes emerged based on the participants' qualitative feedback included family involvement, calendar portability and calendar design., Conclusions: The findings suggest the Diabetes Self-Care Calendar is feasible as a reminder tool for type 2 diabetes mellitus patients who live in rural areas., Competing Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (© The Author(s) 2019.)

The study investigated the effect of finisher diets containing two-stage fermented banana peel (FBP) meal on growth performance, haematological responses, intestinal microbiology and carcass traits of broilers. From days 22 to 38, 200 broiler chicks were grouped to maize-soybean meal-based finisher diet as a control (CONT) and finisher diets containing either 5% FBP (FBP-5), 10% FBP (FBP-10) or 15% FBP (FBP-15). Birds were blood sampled and slaughtered at day 38. For microbial population and villi height measurement, digesta and intestinal segments were collected. Breast meat was obtained for meat colour determination. Data collected were analysed according to analysis of variance followed by Duncan's multiple-range test when there was significant effect. There was no effect of treatments on broiler growth performance. The relative duodenum weight tended (P = 0.08) to be lower in FBP than in CONT. The mean corpuscular haemoglobin concentration (MCHC) was higher (P < 0.05) in FBP-5 than in CONT and FBP-15. Uric acid concentration was lower (P < 0.05) in FBP-10 and FBP-15 than in CONT. Feeding FBP tended (P = 0.08) to decrease coliform population in the ileum. Compared to CONT, the ileal villi heights were higher (P < 0.05) and lower (P < 0.05) in FBP-10 and FBP-5, respectively. The relative weight of thigh (P < 0.05) and wings (P = 0.07) were higher in CONT than in FBP-fed birds, but the eviscerated carcass was not different (P > 0.05) among groups. The lightness (L*) values of meat were higher (P < 0.05) in FBP-10 and FBP-15 than in CONT and FBP-5. Compared with others, breast meat from FBP-10 birds had lower (P < 0.05) redness (a*) values. In conclusion, dietary inclusion of FBP at the levels of up to 15% had no detrimental effect on growth and health performances of broiler chickens.

Background: Diabetes complications can increase progressively, leading to morbidity and mortality among diabetes sufferers. To address this, diabetes self-management education is a strategy to increase patients' knowledge and awareness in self-care. However some issues related to diabetes self-management education have emerged for health care providers and patients., Objectives: The objective of this project was to improve nurses' skills and confidence in delivering diabetes self-management education among type 2 diabetes patients by promoting evidence based practice in community health centers, thereby enhancing patients' knowledge and skills in performing self-care., Methods: The Joanna Briggs Institute three-phase Practical Application of Clinical Evidence System and Getting Research into Practice audit and feedback tool were utilized in this project for promoting evidence utilization and changes in the community health setting. In phase 1 of the project, stakeholders were engaged and 12 evidence-based audit criteria were developed. A baseline audit was conducted by the project team in 41 community health centers. In phase 2, barriers underpinning areas of noncompliance found in the baseline audit were identified and strategies developed to target three key areas - staff, administration and patients. In phase 3, a follow-up audit was conducted., Results: The baseline audit results showed that eight of the 12 criteria recorded zero percent compliance, with the remaining four recording below 50%, representing poor compliance with the current evidence. Strategies implemented during phase 2 of the project included a diabetes educator training program, development of diabetes education curriculum and media, and a patient's logbook. After implementation of the strategies, the follow-up audit showed that all 12 criteria achieved 100% compliance., Conclusions: The diabetes training program for community health center nurses increased their skills and confidence in delivering diabetes self-management education to patients.

The aim of this study was to characterise the gut microbiota composition of piglets fed bovine colostrum (BC), milk replacer (MR) or sow milk (SM) in the post-weaning period. Piglets (n 36), 23-d old, were randomly allocated to the three diets. Faecal samples were collected at 23, 25, 27 and 30 d of age. Digesta from the stomach, ileum, caecum and mid-colon was collected at 30 d of age. Bacterial DNA from all samples was subjected to amplicon sequencing of the 16S rRNA gene. Bacterial enumerations by culture and SCFA analysis were conducted as well. BC-piglets had the highest abundance of Lactococcus in the stomach (P<0·0001) and ileal (P<0·0001) digesta, whereas SM-piglets had the highest abundance of Lactobacillus in the stomach digesta (P<0·0001). MR-piglets had a high abundance of Enterobacteriaceae in the ileal digesta (P<0·0001) and a higher number of haemolytic bacteria in ileal (P=0·0002) and mid-colon (P=0·001) digesta than SM-piglets. BC-piglets showed the highest colonic concentration of iso-butyric and iso-valeric acid (P=0·02). Sequencing and culture showed that MR-piglets were colonised by a higher number of Enterobacteriaceae, whereas the gut microbiota of BC-piglets was characterised by a change in lactic acid bacteria genera when compared with SM-piglets. We conclude that especially the ileal microbiota of BC-piglets had a closer resemblance to that of SM-piglets in regard to the abundance of potential enteric pathogens than did MR-piglets. The results indicate that BC may be a useful substitute for regular milk replacers, and as a feeding supplement in the immediate post-weaning period.

The effect of feeding fermented dried cassava (gathot) on the performances and haematological profile of broilers was investigated. There were four dietary treatments arranged in a completely randomized design, i.e. control diet and diets containing 25, 50 or 100 g/kg gathot. The birds were provided with the treatment diets ad libitum from 8 to 35 days of age. Body weight, feed intake and feed conversion ratio (FCR) were determined weekly. At day 32, the birds were blood sampled, sacrificed and immediately the internal organs and abdominal fat were removed and weighed. Feeding gathot at various levels did not affect (P > 0.05) the growth and FCR, but tended (P = 0.09) to reduce the feed cost per kilogramme live weight gain of broilers. The dietary treatments did not cause toxicological effect on broilers, indicated by the values of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) that were not different from those in the control diet. Dietary inclusion of gathot lowered heterophils to lymphocytes ratio (H/L ratio) (P < 0.05) and albumin to globulin ratio (A/G ratio) (P = 0.14) of broilers as compared to the control diet. Total triglyceride was lower (P < 0.05) in the serum of broilers fed diets containing 5 and 10 % of gathot, when compared with that in the control diet. The treatments resulted in reduced abdominal fat deposition in broilers. In conclusion, dietary inclusion of gathot at up to 10 % had no negative impact on the growth performance of broilers. Feeding gathot has potential to improve the health and physiological stress responses as well as reduce body fat deposition in broilers.

The study aimed to evaluate the probiotic properties, antioxidant activity and fermentative capacity of Acremonium charticola and Rhizopus oryzae isolated from the Indonesian fermented dried cassava, with particular application on poultry. A. charticola inhibited the growth of Escherichia coli and Aspergillus flavus. A. charticola and R. oryzae grew in potato dextrose agar (PDA) adjusted to pH 3 and 8 or in PDA supplemented with bile salt up to 0.8%. After soaking for 8 hr, the survival rate of A. charticola in the simulated gastric juice (pH 2) and bile solutions (2% bile salt) was lower than that of R. oryzae. A. charticola and R. oryzae exhibited strong antioxidant activities. Compared to unfermented cassava pulp (control), the fibre content of cassava pulp tended to be lower after fermentation with A. charticola for 14 days. The populations of A. charticola and R. oryzae were significantly higher in fermented cassava pulp than in unfermented one. Coliform was higher in cassava pulp fermented with R. oryzae or A. charticola + R. oryzae compared to control after 7 days of fermentation, however, the bacteria were not different between A. charticola-fermented cassava pulp and control. Lactic acid bacteria (LAB) were higher in A. charticola- and R. oryzae-fermented cassava pulp than those in control, however, no difference of LAB was observed between A. charticola + R. oryzae-fermented cassava pulp and control. In conclusion, A. charticola exhibited antibacterial, antifungal and antioxidant activity, gastrointestinal persistence and fermentative capacity that may be beneficial for poultry industry.

The present study investigated the effect of feeding bovine colostrum (BC) to piglets in comparison with feeding a milk replacer (MR) and conventional rearing by the sow on the intestinal immune system and number of enterotoxigenic Escherichia coli (ETEC) colonising the intestinal tissue. Piglets (23-d-old) were allocated to one of the following four groups: (1) killed at the beginning of the experiment (Base); (2) separated from the sow and fed BC (BC-fed); (3) separated from the sow and fed a MR (MR-fed); (4) kept with the sow (Sow-Milk). Blood was sampled on days 1 and 8, and faecal samples were collected on days 1, 3, 5 and 8. On day 8, piglets were killed and gastrointestinal digesta and intestinal segments were collected. The frequency of diarrhoea was found to be higher (P≤ 0·019) in MR-fed piglets than in BC-fed and Sow-Milk piglets. Piglets from the MR-fed group had the lowest lactic acid bacteria:haemolytic E. coli ratio (P(treat)= 0·064) in the faeces. The number of E. coli colonising the intestinal tissue was higher (P< 0·001) in piglets from the MR-fed group than in those from the BC-fed and Sow-Milk groups. Piglets from the Sow-Milk group had a higher (P= 0·020) mucosal IgG concentration than those from the MR-fed group, but did not exhibit any difference when compared with piglets from the Base and BC-fed groups. Piglets from the BC-fed group exhibited a reduced (P≤ 0·037) expression level of Toll-like receptor-4 in the intestinal mucosa when compared with those from the MR-fed and Sow-Milk groups. The expression level of IL-2 was higher (P≤ 0·051) in piglets from the MR-fed group than in those from the other treatment groups. In conclusion, feeding BC rather than MR to the piglets reduced the colonisation of intestine by ETEC and modulated the intestinal immune system, whereas no differences were observed in piglets fed BC and conventionally reared by the sows.

Background: The processes of weaning and exposure to pathogenic bacteria induce stress responses, which may alter the metabolism. In this study, we investigated the changes in plasma metabolites and immune responses in piglets in response to the stress induced by weaning and Escherichia coli challenge., Results: Fecal dry matter decreased (P = 0.003) and nearly half (44.4%) the piglets developed diarrhea on day 2 and 3 postweaning. The concentration of plasma immunoglobulin A was higher (P < 0.001) on day 11 postweaning than on day 0 or 4 postweaning. The levels of white blood cells increased continuously (P < 0.001) from day 0 to day 11 postweaning. Differences in the percentages of neutrophils (P = 0.029) and lymphocytes (P = 0.022) were seen, but the neutrophil/lymphocyte ratio did not differ in the period after weaning. A clear separation of the metabolomic profile data for day 0 and day 4 postweaning was observed with a principal components analysis (PCA) scores plot, and the data for day 11 were located between those for day 0 and day 4 postweaning. The plasma levels of proline, taurine, and carnitine were higher, whereas those of betaine, creatine, L-arginine and acetylcarnitine were lower on day 4 postweaning than on day 0. Levels of lysophosphatidylcholine and phosphatidylcholine were either higher or lower after weaning, depending on the chain lengths or characters of these metabolites., Conclusions: Our results show a clear separation in the plasma metabolomic profiles of piglets that corresponded to the fecal responses to stress on the piglets induced by weaning or exposure to a pathogen (E. coli). These plasma metabolite profiles suggest that the challenges induced proinflammatory responses in the piglets, resulting in postweaning diarrhea, which was associated with higher concentrations of IgA in the plasma.

Although the main responsibility for informed consent of medical procedures rests with doctors, nurses' roles are also important, especially as patient advocates. Nurses' preparation for this role in settings with a hierarchical and communal culture has received little attention. We explored the views of hospital managers and nurses regarding the roles of nurses in informed consent and factors influencing these roles. We conducted a qualitative study in a private, multispecialty hospital in Indonesia. Semi-structured interviews were conducted with seven managers. Two rounds of focus group discussions with nurses (n = 27) were conducted. Constant comparative approach was used in the analysis. Nurses can act as manager, witness, information giver, and advocate in the informed consent process. These roles are influenced by nurses' preparedness, hospital culture and policy, patients' understanding, family involvement, and cost-related issues. In preparation for these tasks, nurses should acquire communication skills, clinical knowledge, and legal and ethical knowledge.

Enterotoxigenic Escherichia coli (ETEC) F4 is a major cause of diarrhea in both neonatal and young pigs. Indeed, only pigs having F4 receptors are susceptible. Among the susceptible pigs, it is yet unknown if spontaneous E. coli postweaning diarrhea (PWD) occurrence and intestinal mucosal responses to ETEC differ between genotypes. This study investigated a diarrhea-like condition and intestinal mucosal responses in F4 homo- and heterozygous susceptible weaner pigs. Sixteen weaned pigs (28 d of age) were used in a 2 × 2 factorial study with genotype (homo- or heterozygous F4R(+)) and inoculation with E. coli F4 or not as the 2 factors. Within genotype, 4 pigs were inoculated with E. coli F4 and the other 4 pigs received saline buffer on days 7 and 8 after weaning. Fecal score and DM and bacterial counts were conducted from days 7 to 12 after weaning. Blood was obtained on days 3 and 10 after weaning and at the time of killing. Four pigs were killed per day on days 14, 15, 16, and 17. Small intestine (SI) was divided into 3 parts of equal length for measurement of intestinal weight and the amount of mucosa. Lymphocyte subsets in jejunal Peyer's patches (jejPP) were analyzed using flow cytometry. Escherichia coli reduced (P = 0.05) total percentage of intestinal mucosa (on a dry basis) and had an impact on metabolomics profile of the plasma. No effect of genotype was seen on fecal score and DM, fecal shedding of hemolytic E. coli, mucosal responses, metabolomics profile, antibody responses, and lymphocyte subsets counts. This study suggests that both F4 homo- and heterozygous susceptible pigs have similar functional receptors for E. coli F4, which facilitate the adhesion of F4 to the intestinal tissue.

Ligand-gated ion channels efficiently couple neurotransmitter binding to the opening of an intrinsic ion channel to generate the post-synaptic potentials that are characteristic of fast synaptic transmission. In the Cys-loop family of ligand-gated ion channels, the ligand-binding site is approximately 60 Å above the channel gate. Structural modelling of related proteins and mutagenesis studies led to the hypothesis that loops 2 and 7 of the extracellular domain may couple ligand binding to receptor activation. Mutating loop 2 residues of the glycine receptor to cysteine reveals an alternating pattern of effect upon receptor function. Mutations A52C, T54C and M56C produced a threefold right-shift in EC(50) . In contrast, a 30-fold right-shift was seen for mutations E53C, T55C and D57C. Loop 2 conformational changes associated with ligand binding were assessed by measuring the rate of covalent modification of substituted cysteines by charged methane thiosulfonate reagents. We show for the first time state-dependent differences in the rate of reaction. A52C and T54C are more accessible in the resting state and M56C is more accessible in the activated state. These results demonstrate that loop 2 does undergo a conformational change as part of the mechanism that couples ligand binding to channel opening., (© 2010 The Authors. Journal of Neurochemistry © 2010 International Society for Neurochemistry.)

The functional role of ligand-gated ion channels in the central nervous system depends on their relative anion-cation permeability. Using standard whole-cell patch clamp measurements and NaCl dilution potential measurements, we explored the effect of external divalent ions on anion-cation selectivity in alpha1-homomeric wild-type glycine receptor channels. We show that increasing external Ca(2+) from 0 to 4 mM resulted in a sigmoidal increase in anion-cation permeability by 37%, reaching a maximum above about 2 mM. Our accurate quantification of this effect required rigorous correction for liquid junction potentials (LJPs) using ion activities, and allowing for an initial offset potential. Failure to do this results in a considerable overestimation of the Ca(2+)-induced increase in anion-cation permeability by almost three-fold at 4 mM external Ca(2+). Calculations of LJPs (using activities)&#95; were validated by precise agreement with direct experimental measurements. External SO (4) (2-) was found to decrease anion-cation permeability. Single-channel conductance measurements indicated that external Ca(2+) both decreased Na(+) permeability and increased Cl(-) permeability. There was no evidence of Ca(2+) changing channel pore diameter. Theoretical modeling indicates that the effect is not surface charge related. Rather, we propose that, under dilution conditions, the presence of an impermeant Ca(2+) ion in the channel pore region just external to the selectivity filter tends to electrostatically retard outward movement of Na(+) ions and to enhance movement of Cl(-) ions down their energy gradients.

The functional role of ion channels, which allow counterion permeation, depends critically on their relative anion-cation relative selectivity. From whole-cell patch clamp reversal potential measurements under dilution potential conditions, we have already shown that anion-cation permeabilities of anion-selective wild-type (WT) and mutant (with larger pore diameter) glycine receptor (GlyR) channels in the presence of Li(+), Na(+) and Cs(+) counterions, were inversely correlated with the equivalent hydration diameter of the counterion, with chloride-cation permeability increasing as counterion equivalent hydration diameter increased with respect to the channel minimum pore diameter. Corrected for liquid junction potentials (LJPs; using ion activities), the previous chloride-cation permeabilities for the alkali cations were 23.4 (Li(+)), 10.9 (Na(+)) and 5.0 (Cs(+)) for the smaller WT channel. Further analysis to incorporate an initial offset potential correction, to fully allow for slight differences between internal cell composition and external control salt solution, changed the above permeability ratios to 30.6 (Li(+)), 11.8 (Na(+)) and 5.0 (Cs(+)), adding enhanced support for the inverse correlation between anion-to-counterion permeability ratio and equivalent hydrated counterion diameter relative to channel pore diameter (erroneously ignoring LJPs reduces each permeability ratio to about 4). Also, new direct measurements of LJPs (for NaCl and LiCl salt dilutions) using a 3M KCl-agar reference salt bridge (with freshly-cut end for each solution composition change) have shown excellent agreement with calculated LJPs (using ion activities), validating calculated LJP values. We continue to suggest that counterion cations permeate with chloride ions as neutral pairs.

The Cys loop receptor channels mediate fast synaptic transmission in the nervous system. The M2-demarcated transmembrane pore is an important determinant of their ion permeation properties. Portals within the intracellular domain are also part of the permeation pathway in cationic Cys loop receptors, with charged residues in a helical MA stretch partially lining these openings profoundly affecting channel conductance. It is unknown whether analogous portals contribute to the permeation pathway in anionic Cys loop receptors. We therefore investigated the influence of charged residues within the proposed MA stretch on functional properties of the homomeric glycine alpha1 receptor. Up to eight basic residues in the MA stretch were concurrently mutated to a negatively charged glutamate, and wild-type and mutant subunits were expressed in HEK-293 cells. Mutation of all eight residues produced a non-functional receptor. The greatest reduction in conductance at negative membrane potentials (from 92.2+/-2.8 to 60.0+/-2.2 picosiemens) was observed with glutamate present at the 377, 378, 385, and 386 positions (the 4E subunit). Inclusion of additional glutamate residues within this subunit did not decrease conductance further. Neutralizing these residues (the 4A subunit) caused a modest decrease in conductance (80.5+/-2.3 picosiemens). Outward conductance at positive potentials was not markedly affected. Anion to cation selectivity and concentration-response relationships were unaffected by the 4A or 4E mutations. Our results identify basic residues affecting conductance in the glycine receptor, suggesting that portals are part of the extended permeation pathway but that the M2-demarcated channel pore is the dominant determinant of permeation properties in glycine receptors.

The functional role of ligand-gated ion channels depends critically on whether they are predominantly permeable to cations or anions. However, these, and other ion channels, are not perfectly selective, allowing some counterions to also permeate. To address the mechanisms by which such counterion permeation occurs, we measured the anion-cation permeabilities of different alkali cations, Li(+) Na(+), and Cs(+), relative to either Cl(-) or NO(3)(-) anions in both a wild-type glycine receptor channel (GlyR) and a mutant GlyR with a wider pore diameter. We hypothesized and showed that counterion permeation in anionic channels correlated inversely with an equivalent or effective hydrated size of the cation relative to the channel pore radius, with larger counterion permeabilities being observed in the wider pore channel. We also showed that the anion component of conductance was independent of the nature of the cation. We suggest that anions and counterion cations can permeate through the pore as neutral ion pairs, to allow the cations to overcome the large energy barriers resulting from the positively charged selectivity filter in small GlyR channels, with the permeability of such ion pairs being dependent on the effective hydrated diameter of the ion pair relative to the pore diameter.