B. pseudomallei. In general, soil specimens are cultured in enrichment broth for 2 days, and then the culture broth is streaked on an agar plate and incubated further for 7 days. However, identifying B. pseudomallei on the agar plates among other soil microbes requires expertise and experience. Here, we evaluate a lateral flow immunoassay (LFI) developed to detect B. pseudomallei capsular polysaccharide (CPS) in clinical samples as a tool to detect B. pseudomallei in environmental samples.Culture is the gold standard for the detection of environmental B. pseudomallei were spiked with B. pseudomallei ranging from 10 to 105 CFU, and incubated in 10 ml of enrichment broth in air at 40°C. Then, on day 2, 4 and 7 of incubation, 50 μL of the upper layer of the broth were tested on the LFI, and colony counts to determine quantity of B. pseudomallei in the broth were performed. We found that all five soil specimens inoculated at 10 CFU were negative by LFI on day 2, but four of those five specimens were LFI positive on day 7. The LOD of the LFI was estimated to be roughly 3.8x106 CFU/ml, and culture broth on day 7 was selected as the optimal sample for LFI testing. Second, we evaluated the utility of the LFI by testing 105 soil samples from Northeast Thailand. All samples were also tested by standard culture and quantitative PCR (qPCR) targeting orf2. Of 105 soil samples, 35 (33%) were LFI positive, 25 (24%) were culture positive for B. pseudomallei, and 79 (75%) were qPCR positive. Of 11 LFI positive but standard culture negative specimens, six were confirmed by having the enrichment broth on day 7 culture positive for B. pseudomallei, and an additional three by qPCR. The LFI had 97% (30/31) sensitivity to detect soil specimens culture positive for B. pseudomallei.First, we determined the limit of detection (LOD) of LFI for enrichment broth of the soil specimens. Soil specimens (10 grams/specimen) culture negative for B. pseudomallei in soil samples, and to select which samples should be sent to reference laboratories or proceed further for bacterial isolation and confirmation. This could considerably decrease laboratory workload and assist the development of a risk map for melioidosis in resource-limited settings.The LFI can be used to detect  Burkholderia pseudomallei is an environmental Gram-negative bacillus and the causative agent of melioidosis. Culture and PCR assays are standard diagnostic tools used to detect B. pseudomallei in the environment. However, those tests require experienced microbiologists and are regularly conducted only in a few research laboratories worldwide. In this study, we demonstrated that the prototype lateral flow immunoassay (LFI) developed to detect B. pseudomallei capsular polysaccharide (CPS) in clinical samples could be used to detect B. pseudomallei in environmental samples. We found that the LFI can be used to detect B. pseudomallei in experimentally spiked soil specimens. Next, we evaluated the sensitivity of LFI using 105 soil samples collected in Northeast Thailand. We found that the LFI had high sensitivity to detect B. pseudomallei in the soil. We propose that the LFI could be used to detect environmental B. pseudomallei in resource-limited settings. Soil samples positive for LFI could be sent to reference laboratories for confirmation with culture or molecular methods. The use of LFI could assist in the development of a global risk map for melioidosis. Burkholderia pseudomallei is a soil-dwelling organism and also the cause of melioidosis, an often fatal infectious disease [B. pseudomallei may be dismissed as a culture contaminant [B. pseudomallei [B. pseudomallei is likely present in a further 34 countries in which melioidosis has never been reported [The Gram-negative  disease ,2. Melio disease . Even witaminant , or be mtaminant ,6. The ddomallei . The orgdomallei ,9. A recdomallei . The stureported .B. pseudomallei in countries where B. pseudomallei is likely present but melioidosis has never been reported is important, since this will provide policy makers with evidence for raising awareness of this disease among healthcare workers and microbiology laboratories in these areas [B. pseudomallei prior to the recognition of human disease [B. pseudomallei from clinical specimens, with the first case of melioidosis being identified in 1999 [B. pseudomallei after case reports in Gabon [Defining the distribution of se areas . Environ disease . This en in 1999 . Since t in 1999 . Environin Gabon  and Bangin Gabon .B. pseudomallei in the environment, but both tests require experienced microbiologists and are only available in a few research laboratories worldwide. For the culture method, soil specimens are initially cultured in selective broth for 2 days, and then the upper layer of the broth is streaked on an agar plate and incubated for a further 7 days [B. pseudomallei on agar plates among other soil microbes is time-consuming and requires expertise and experience. For effective molecular detection, soil specimens are also first enriched in selective broth for 2 days prior to nucleic acid amplification [Culture and PCR assays are commonly used to detect r 7 days . Identiffication ,18. DespB. pseudomallei capsular polysaccharide (CPS) in clinical samples [B. pseudomallei greater than 1.2×104 CFU/ml [B. pseudomallei in the enrichment broth of the environmental samples. We found that the LFI had high sensitivity in our setting.Recently, a lateral flow immunoassay (LFI) was developed to detect  samples . In a pi4 CFU/ml . Here, wB. pseudomallei wild-type strain E08 and 300 grams of soil culture negative for B. pseudomallei collected from Nakorn Ratchasima, Northeast Thailand, were used. The strain E08 was selected as the strain has been frequently used as a representative strain of B. pseudomallei from the environment [B. pseudomallei at an inoculum of 10 to 105 colony forming units (CFU) per 10 grams of soil, and incubated in 10 ml of enrichment broth  in air at 40°C. Unspiked soil samples and soil samples sterilised by autoclaving were included as negative controls. Soil samples sterilised by autoclaving, TBSS-C50 and sterile distilled water, all spiked with B. pseudomallei, were also included as positive controls. After incubation for 2, 4 and 7 days, the upper layer of enrichment broth were tested by LFI, and evaluated by culture for quantity of B. pseudomallei. A total of nine soil specimens were used to cover the range of inoculum from 10 to 105 CFU per 10 grams of soil, while five specimens was used for the lowest inoculum of 10 CFU per 10 grams of soil  was sprayed on the same membrane for the control line. The conjugate pad contained dried 40 nm gold particles conjugated with mAb 4C4 as well as a small amount of gold conjugated with chicken IgY (to react with the control line). The conjugate pad was treated with a borate-based buffer containing a small concentration of detergent and dried for later gold conjugate application. The sample application pad was also treated similarly and dried. The LFI was assembled by combining the sprayed membrane, conjugate pad, and sample pad on top of an adhesive plastic backing. The LFI was stored in air at room temperature until use. A total of 50 μl of the upper layer of the broth was mixed with two drops of lysis buffer and two drops of chase buffer in a clean tube, in which the LFI pad was dipped. After 15 minutes at room temperature, the LFI results were read.The Active Melioidosis Detect (AMD) LFI developed by InBios  was used . The evaB. pseudomallei in the enrichment broth was performed as described previously with minor modifications [B. pseudomallei was identified as previously described [B. pseudomallei results between samples with positive and negative LFI results. The quantitative counts of B. pseudomallei in specimens with high levels of fungus overgrowth were excluded. The optimal duration of enrichment for LFI testing was determined by selecting the duration that provided the highest proportion of LFI positivity.The quantitative count of ications . In shorescribed . The LODB. pseudomallei developed by DEBWorP [B. pseudomallei was then identified as previously described [B. pseudomallei in soil specimens which were negative by standard culture but LFI positive. Three microbiologists performed culture, LFI and qPCR independently, and they were blinded to the results of the other two tests.A total of 105 environmental soil samples were collected from 21 rice paddy fields located in 7 provinces  in Northeast Thailand during March and June 2015. Three villages per each province were randomly selected for study. We calculated that 105 samples were needed to estimate sensitivity of LFI with the 95% confidence interval of 15%. The randomization was performed using Stata version 14.0 . A rice field in each village was selected, and the first 5 soil samples collected in each rice field were used for the study. In each rice field, the soil samples was collected 2.5 meters apart. The soil sampling and culture was performed according to the consensus guidelines for environmental sampling of  DEBWorP  with sli DEBWorP , and B. escribed . In addiorf2 of B. pseudomallei type III secretion system (TTS1) was performed as previously described [A 1 mL aliquot of broth on day 7 was stored at -20°C until further processing. Prior to nucleic acid extraction, the broth was spun at 12,000g for 10 min, and all but the pellet and 200 μL of broth were removed. The remaining DNA pellet and broth were processed using the Qiagen Mini Kit using the bacterial extraction protocol, according to the manufacturers instructions. The qPCR assay targeting escribed .B. pseudomallei in soil samples [P value. This describes the level of association, both positive and negative, beyond that caused by chance, as follows: 0.00–0.20, slight; 0.21–0.40, fair; 0.41–0.60, moderate; 0.61–0.80, substantial; 0.81–1.00, high.Utility of the LFI was determined by comparing the results between culture of broth on day 2, LFI of broth on day 7 and qPCR of broth on day 7. Culture of broth on day 2 was selected for the main comparison because it is the standard method used for environmental detection of  samples . The degB. pseudomallei at an inoculum of 10 to 105 CFU per 10 grams of soil, and incubated in 10 ml of enrichment broth in air at 40°C  were spiked with  at 40°C . Immedia of soil . The 100B. pseudomallei in the broth of five soil specimens spiked with 10 CFU ranged from 3x103 to 8.4x105 CFU/ml. Nonetheless, all of those five specimens were LFI negative  was LFI positive, while the remaining four were LFI negative. On day 7, B. pseudomallei count in the broth of those five specimens ranged from 9x103 to 1.3x108 CFU/ml. There was some difficulty obtaining quantitative counts in the two specimens with low counts (9x103 and 2x104 CFU/ml) due to overgrowth with fungi. Nonetheless, four specimens were LFI positive, while the remaining one with the count of 2x104 CFU/ml was LFI negative , and the next higher count that had LFI positive was 3.8x106 CFU/ml  were LFI positive, and 79 (75%) were qPCR positive  sensitivity for detection of soil specimens culture positive for B. pseudomallei.Of 105 soil samples collected in Northeast Thailand, 25 (24%) were culture positive for positive . AgreemeB. pseudomallei in soil samples. The LFI has a high sensitivity (97%) to detect soil specimens culture positive for B. pseudomallei. The test is very simple to use and does not require expertise or new equipment; therefore, LFI could be useful for inexperienced microbiologists who want to conduct an environmental survey in resource-limited settings. One explanation of the high sensitivity of the LFI is that we chose the enriched broth on day 7 as the specimen for LFI. Enrichment of soil specimens increases the concentration of B. pseudomallei in the broth exponentially, and the enrichment step (for 1–2 days) is already necessary for both culture and molecular detection [B. pseudomallei in the broth from as low as 1 CFU/ml  up to 3.8 x 106 CFU/ml high enough to be detected.This study demonstrates the utility of LFI as a tool to detect etection ,18. We sB. pseudomallei, it requires expertise and experience. Colonies of B. pseudomallei on Ashdown agar plates can have seven morphologies [B. pseudomallei in soil samples as the reading is simple and can be performed by microbiologists otherwise not experienced with B. pseudomallei. Preliminary detection of B. pseudomallei in enrichment media of soil samples by LFI will also reduce the workload of at melioidosis reference laboratories by focusing on the confirmation of identified LFI positives rather than screening large sample numbers. This could enhance the development of a global risk map for melioidosis, particularly in areas without melioidosis reference laboratories [Although culture is the gold standard for the detection of environmental hologies  and be dhologies . In addihologies , particuratories .6 CFU/ml) is comparatively higher than its previously reported LOD in direct urine samples (1.2 x 104 CFU/ml) [The LOD of LFI in soil enrichment (3.8x10 CFU/ml) . This isB. pseudomallei can survive well in soil kept at ambient temperature (24 to 32°C) and away from direct sunlight [B. pseudomallei in soil increased moderately over 2 weeks when kept at 20°C [B. pseudomallei provides definitive evidence that people in the areas sampled are at risk of melioidosis; therefore, it is important to use the test or a combination of tests with high accuracy rather than a rapid test with low accuracy.The long total time required to complete the LFI test (7 days) is not a major problem as the total time required for the culture method is also 9 days. The culture method requires the soil specimen to be in the enrichment broth for 2 days, and then the upper layer of the broth is streaked on plates and incubated for a further 7 days. sunlight . Therefo at 20°C . The isoB. pseudomallei. Second, qPCR has a lower detection limit compared to the culture method [B. pseudomallei [B. pseudomallei in an additional three specimens that were culture negative but LFI positive. As qPCR is more sensitive than culture and LFI, the molecular methods should be considered if absence of the organism in the environment needs to be confirmed and resources are available [The high proportion of specimens with were culture negative but qPCR positive  could bee method , and coudomallei . In thisvailable . NonetheB. thailandensis which produces CPS similar to B. pseudomallei is present in the soil. Second such B. thalandensis variants have been reported in soil from the USA (Texas) [B. pseudomallei in the environment . If transportation of LFI-positive soil samples to reference laboratories is required, it would be preferred within 7-days of completing the LFI tests [B. pseudomallei developed by DEBWorP [B. pseudomallei numbers lower and/or other organisms are present. Fifth, Our study sample size is not large. Further studies should also investigate the sensitivity and utility of the LFI to detect B. pseudomallei in soil outside Northeast Thailand.The LFI may have some limitations. First, the LFI could yield false positive results if a variant of  (Texas) , Cambodi (Texas)  and Laos (Texas) , but notFI tests . Sample  DEBWorP . Nonethe DEBWorP . Fourth,B. pseudomallei in new areas, particularly when the investigation is conducted by microbiologists who have limited experience with the isolation and identification of B. pseudomallei from soil specimens. LFI positive soil specimens could then be sent to reference laboratories for bacterial isolation and confirmation. This could considerably decrease laboratory workload and assist in the development of a global risk map for melioidosis.In conclusion, we recommend that the LFI could be used to detect environmental S1 Table(DOCX)Click here for additional data file.


Sinorhizobium fredii is a fast-growing rhizobial species that can establish a nitrogen-fixing symbiosis with a wide range of legume species including soybeans (Glycine max). In soybeans, this interaction shows a high level of specificity such that particular S. fredii strains nodulate only a limited set of plant genotypes. Here we report the identification of a dominant gene in soybeans that restricts nodulation with S. fredii USDA193. Genetic mapping in an F2 population revealed co-segregation of the underlying locus with the previously cloned Rfg1 gene. The Rfg1 allele encodes a member of the Toll-interleukin receptor/nucleotide-binding site/leucine-rich repeat class of plant resistance proteins that restricts nodulation by S. fredii strains USDA257 and USDA205, and an allelic variant of this gene also restricts nodulation by Bradyrhizobium japonicum USDA122. By means of complementation tests and CRISPR/Cas9-mediated gene knockouts, we demonstrate that the Rfg1 allele also is responsible for resistance to nodulation by S. fredii USDA193. Therefore, the Rfg1 allele likely provides broad-spectrum resistance to nodulation by many S. fredii and B. japonicum strains in soybeans. The leguminous plants are able to establish a symbiotic relationship with nitrogen-fixing soil bacteria called rhizobia. The symbiosis is featured by the formation of root nodules where the bacteria in nodule cells can convert atmospheric nitrogen into ammonia and make it available to the plant. This symbiotic partnership has important implications in sustainable agriculture because it reduces the need for nitrogen-based fertilizers.Glycine max) and alfalfa (Medicago sativa) is through root hairs. Bacteria are first entrapped by curled root hairs and multiply to form micro-colonies referred to as infection foci. From these foci, plant-made tubular-like structures, called infection threads, start to develop through local cell wall hydrolysis and invagination of the plant plasma membrane. The infection threads that are colonized by dividing bacteria proceed through the epidermal cell layer into the inner cortex where the nodule primordium has formed. Bacteria are then internalized in an endocytosis-like process and surrounded by a host membrane, where they differentiate into nitrogen-fixing bacteroides and are confined in organelle-like structures called symbiosomes  factors . Recognibiosomes .While some bacteria can nodulate a wide range of hosts, most bacteria have strict host selectivity. As such, particular rhizobial species or strains nodulate only a narrow group of legume species or genotypes . UnderstEstablishment of a root nodule symbiosis requires mutual recognition of multiple molecular signals between the symbiotic partners . TherefoRj2, Rfg1, and Rj4) that restrict nodulation with specific rhizobial strains  resistance protein that confers resistance to nodulation by specific strains of Bradyrhizobium japonicum and Sinorhizobium fredii, respectively  while Williams 82 restricted nodulation by this strain (Nod-) . The strain was cultured on YEM agar plates  in the dark at 28°C for 4–5 days, and the bacterial paste was then collected and diluted in sterile water to OD600 of 0.1. For nodulation assay, each 1-week-old seedling was flood-inoculated with 10 mL of the bacterial suspension. Plants were grown in sterilized 50/50 Perlite-Turface mix in a growth chamber programmed for 16 h light at 26°C and 8 h dark at 23°C. Nodulation phenotype was assayed 4 weeks post inoculation. Genotyping was conducted by using a CAPS (cleaved amplified polymorphic sequences) marker developed based on a SNP (single nucleotide polymorphism) between the Rfg1 (Williams 82) and rfg1 (Peking) alleles. The primer pair used was 5′TGAGAGTACTGGAATGGTGGAG3′ and 5′TTGCTGATCGAACCACTCTG3′, and the restriction enzyme used was HpaI.The F2 mapping population was derived by crossing the North American cultivar ‘Williams 82’ with the cultivar ‘Peking,’ an introduction from China. Peking formed nitrogen-fixing nodules when inoculated with n (Nod-) . S. fredom China , was obtRfg1 was used for complementation tests. Genomic DNA of the Rfg1 allele was derived from the BAC clone Gm_WBa0019D20 of Williams 82  background by means of hairy root transformation. To validate the CRISPR/Cas9-mediated gene disruption, the roots that formed nodules were subjected to DNA isolation, PCR amplification, and DNA sequencing. If the initial sequencing suggested the presence of multiple heterogeneous mutant alleles, the PCR product was ligated into pGEM T-Easy Vector System (Promega) and at least 10 colonies were selected for sequencing.The CRISPR/Cas9 gene knockout constructs were developed based on the pHSE401 vector described by Rfg1 allele that restricts nodulation by S. fredii USDA257 is allelic to Rj2, an allele that restricts nodulation by B. japonicum USDA122 (Rj2 (rfg1) allele that restricts nodulation with USDA122 but allows nodulation with USDA257, the rj2 (Rfg1) allele that permits nodulation with USDA122 but restricts nodulation with USDA257, and the rj2 (rfg1) allele that nodulates with both strains. These allelic specificities are defined by seven amino-acid substitutions (Rj2 (Rfg1) allele type that prohibits nodulation with both USDA122 and USDA257 in the surveyed soybean lines . We tested the function of this chimeric allele by transferring it into Peking, a genotype that formed nodules with both USDA122 and USDA257. To avoid redundancy, we will provide a further description of this experiment in the “Results” section.The  USDA122 . At thisitutions . Howeveran lines . We thusAgrobacterium rhizogenes-mediated hairy root transformation was carried out based on the protocol described by A. rhizogenes strain K599 that contains individual binary vectors was injected into the cotyledonary node of 1-week-old seedlings using a latex free syringe with a thin needle (0.4 mm × 13 mm) . The infected seedlings were grown in sterile vermiculite and covered with plastic bags in a growth chamber to maintain high humidity. Two to three weeks after inoculation, when hairy roots were well developed at the infection sites, the main roots were removed, and the composite plants were inoculated with rhizobia. Nodulation assays were performed 4 weeks post inoculation.Assay for root hair curling followed the method described in Sinorhizobium fredii USDA193 formed mature nitrogen-fixing nodules on the roots of Peking  but not on the roots of Williams 82 . Despite being unable to form functional nodules in the Williams 82 background, the bacterial strain could frequently induce the formation of rudimentary nodules, bump-like small cortical proliferations on the roots . In contrast to the infected nodules formed in the compatible interaction , the rudimentary nodules on the roots of Williams 82 were completely devoid of infected bacteria; as such, the cortical cell division ceased at very early stage of the nodule development . The ability to produce rudimentary nodules with USDA193 on the Williams 82 roots suggested that the early responses of Nod factor perception are not affected. Consistent with this inference, root hair curling, a hallmark of Nod factor responses, occurred in both compatible and incompatible interactions . Thus, we concluded that the restriction of nodulation by S. fredii USDA193 in Williams 82 was not due to a failure in Nod factor perception but caused by the block of bacterial infection.S. fredii USDA193 in an F2 population derived from the cross between Williams 82 (Nod-) and Peking (Nod+). From a small population of 122 plants inoculated by USDA193, 95 were Nod- and 27 were Nod+. The segregation statistically fits the 3:1 (Nod- to Nod+) ratio , suggesting that the restriction of nodulation by USDA193 in Williams 82 is controlled by a single dominant gene. The dominant nature of the nodulation-restrictive allele supports our hypothesis that the incompatible interaction between Williams 82 and USDA193 was not due to a failure in Nod factor signaling but resembles ‘gene-for-gene’ resistance in the plant–pathogen interactions.We carried out genetic analysis of the symbiosis incompatibility involving S. fredii strains USDA257 and USDA193, and we previously reported that the resistance to nodulation with USDA257 in Williams 82 is controlled by the dominant Rfg1 allele (Glyma16g33780) that encodes a TIR-NBS-LRR protein . For this purpose, we designed two gRNA vectors that individually target two different sites of the fourth exon . The vectors were introduced to A. rhizogenes K599 for hairy root transformation, followed by assaying the nodulation capacity of the hairy roots by inoculation with S. fredii USDA193. For both vectors, we obtained >30 putative transgenic roots from more than 50 independent plants that formed mature nitrogen-fixing nodules. DNA sequencing validated the targeted gene disruption in these roots and these roots did not contain the wild-type allele. Two examples are illustrated in Figures 3C,D. Figure 3C shows that a transgenic root forming nodules resulted from the knockout of Rfg1 at the site 1, and sequence analysis revealed two mutant alleles, one with a 3-bp deletion and another with a 10-bp deletion. Figure 3D represents an example showing that knockout of Rfg1 at the site 2 also led to the formation of root nodules on a transgenic root; sequence analysis identified two mutant alleles, one with a 4-bp deletion and another with a 26-bp deletion when compared with the wild-type allele. Taken together, we conclude that Rfg1 is responsible for nodulation restriction by S. fredii USDA193 in Williams 82.We further used the CRISPR/Cas9-based reverse genetics tool  to knockRfg1 and Rj2 are allelic genes, each encoding a TIR-NBS-LRR protein of 1052 amino acids (Rj2 (rfg1), rj2 (Rfg1), and rj2 (rfg1) (Rj2 (rfg1) allele restricts nodulation with USDA122 but not with USDA257; the rj2 (Rfg1) allele restricts nodulation with USDA257 but not with USDA122; and the rj2 (rfg1) allele allows nodulation with both strains. These allelic specificities are determined by seven amino acid substitutions occurring around the C-terminus of the NBS domain and the sixth LRR repeat . Comparing between Rj2 (rfg1) and rj2 (rfg1) and between rj2 (Rfg1), and rj2 (rfg1) suggests that E452 and I490 are required for the Rj2-mediated nodulation restriction against USDA122, while E731, N736, S743, D756, and S758 are essential for Rfg1-mediated resistance against USDA257 and USDA193. If this inference is true, then the chimeric gene of Rj2 and Rfg1, called Rj2 (Rfg1), encoding a protein with E452, I490, E731, N736, S743, D756, and S758, would prevent nodulation with both strains. We generated multiple transgenic roots (from >100 independent plants) expressing the chimeric gene in Peking that carries an rj2 (rfg1) allele and forms nodules with USDA257, USDA193, and USDA122. In consistence with our hypothesis, the transgenic roots restrict nodulation with both USDA257 , USDA193 , and USDA122 . The transgenic roots retained their ability to nodulate with B. japonicum USDA110, a strain that nodulates both Rj2 and Rfg1 genotypes .no acids . A surve2 (rfg1) . The Rj2Rfg1 gene restricts nodulation with the fast-growing S. fredii strains USDA257 and USDA205  has not yet been identified  and rfg1 (0.56) allele frequencies suggests that these alleles has not been subjected to strong natural selection. However, a screen of 197 soybean lines from the Midwestern United States revealed an Rfg1 allele frequency of 0.83, suggesting that the compatibility with S. fredii strains may be eroded during the breeding process, likely due to the narrow genetic basis of germplasms used in the soybean breeding programs  allele likely have derived from the rj2 (rfg1) allele through gain-of-function mutations. The rj2 (rfg1) allelic form is most promiscuous, conferring an unrestricted nodulation phenotype with many strains that are restricted by the Rj2 or Rfg1 alleles. Therefore, the rj2 (rfg1) genotype should be prioritized in soybean breeding programs if the goal is to ensure the cultivar to be able to nodulate with indigenous bacterial strains.The soybean  strains . A survest China . In contountries . The disprograms . The divRj2/Rfg1 locus encodes a TIR-NB-LRR gene with three allelic variants that show differing specificities to yet unknown bacterial effectors. E452 and I490 in or near the C-terminal NBS domain of Rj2 are required for the recognition of the cognate effector from B. japonicum USDA122, while E731, N736, S743, D756, and S758 in the LRR domain of Rfg1 are essential for the recognition of the corresponding effectors secreted by USDA257 and USDA193. This situation is similar to the previous studies showing that both TIR, NBS, and LRR domains could be involved in the determination of resistance specificity (Rj2(Rfg1) allele composed of E452, I490, E731, N736, S743, D756, and S758 could recognize corresponding effectors of all these strains. Such an allele could exist in natural population if intragenic recombination events occur between the Rj2(rfg1) and rj2(Rfg1) alleles. It is possible that these polymorphic sites play a role in direct effector binding or intra- or intermolecular interactions in protein complexes during recognition and signaling. Further analysis of the mechanisms underlying the recognitions mediated by the Rj2/Rfg1 locus awaits the identification of the cognate bacterial effectors.The polymorphic cificity . The chiYF, JL, SL, and HZ: conception and design of the work. YF, JL, QW, SY, and SL: performed the work and analyzed data. HZ wrote the paper.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 reviewer DR and handling Editor declared their shared affiliation, and the handling Editor states that the process nevertheless met the standards of a fair and objective review.


Mitochondria are fundamental for eukaryotic cells as they participate in criticalcatabolic and anabolic pathways. Moreover, mitochondria play a key role in thesignal transduction cascades that precipitate many  regulatedvariants of cellular demise. In this short review, we discuss the differentialimplication of mitochondria in the major forms of regulated cell death. Both prokaryotic and eukaryotic cells succumb to very harsh microenvironmentalconditions in a virtually instantaneous and uncontrollable manner. Such form ofcellular demise, which has been dubbed “accidental cell death” (ACD), reflects themechanical disassembly of cellular constituents exposed to excessive temperatures,shear forces and/or pressures, and does not involve any molecular machinery 2Generally, RCD is activated once adaptive response to stress fail at the cellularlevel, hence constituting a mechanism for the preservation of organismal homeostasis45689Of note, one specific variant of RCD that is known as “programmed cell death” (PCD)is initiated at a predetermined point of a cell’s life, as a part of(post-)embryonic development or the maintenance of tissue homeostasis in the adult11The signal transduction cascades controlling RCD have expanded considerablythroughout evolution, especially 1) once eukaryotic life has been established, and (2) along withthe transition from a purely unicellular state to multicellularity  1213 once euk161718202122232527282930c, somatic  33Intrinsic apoptosis is a form of RCD initiated by perturbations of intracellularhomeostasis that relies on the catalytic activity of the cysteine protease caspase-3(CASP3) 1151617In physiological conditions, CYTC exclusively resides between the outer and the innermitochondrial membrane, where it is loosely associated with the latter as itoperates as an electron shuttle of the respiratory chain L) and myeloidcell leukemia 1 (MCL1), mainly operate by preventing MOMP In line with this notion, several proteins with prominent anti-apoptotic functions,including various other members of the Bcl-2 family like B-cell CLL/lymphoma 2(BCL2) itself, BCL2-like 1  There are at least two distinct mechanisms whereby BCL2-like proteins mediate such aneffect: (1) by physically interacting with BAX and BAK1 and hence preventing theiroligomerization m, which isrequired for ATP synthesis and several other mitochondrial functions) 4042442 (PGE2) Importantly, MOMP drives intrinsic apoptosis not only as it initiates theapoptosome-dependent activation of CASP3 , but also because it entails the immediatedissipation of the mitochondrial transmembrane potential  get stabilized in the presence of FAS ligand (FASLG),favoring the recruitment of a large multiprotein complex at the cytosolic tail ofthe receptor 51Importantly, distinct death receptors assemble structurally different DISCs uponactivation, implying that the signaling pathway initiated by death receptors canexhibit a remarkable degree of variation  55Necroptosis is a variant of RCD that obligatorily relies on the activation of theRIPK1-like protein receptor-interacting protein kinase 3 (RIPK3) and thepseudokinase mixed lineage kinase domain-like (MLKL), and generally manifests with anecrotic morphology 1152021585961636465-/-Pgam5 mice Initially, mitochondria were thought to participate in necroptotic signaling in atleast two ways: (1) necroptosis was linked to an oxidative burst caused by theRIPK3-dependent activation of various metabolic enzymes, including mitochondrialglutamate dehydrogenase 1 (GLUD1) 68m dissipation, massive water intake, and osmotic organellebreakdown 115222317177374O ATPase  plays acritical function within the PTPC 171The term MPT is commonly employed to indicate an abrupt increase in the permeabilityof the inner mitochondrial membrane to small solutes, resulting in immediateΔψC- (a cystine/glutamate antiporter), resulting inthe depletion of antioxidant defenses and lethal lipid peroxidation 1152577Ferroptosis is an iron-dependent form RCD generally initiated by the inhibition ofplasma membrane system x-/-Ppif cells as wellas in the presence of the MPT inhibitor CsA Of note, Fer-1 and alike fail to inhibit the generation of mitochondrial reactiveoxygen species (ROS) + stores, resultingin a potentially lethal bioenergetic crisis Parthanatos is a peculiar form or RCD depending on poly(ADP-ribose) polymerase 1(PARP1), a nuclear protein involved in DNA repair, and apoptosis inducingfactor, mitochondria associated 1 (AIFM1) 115+/K+ ATPase, implying that it can be modulated withchemical agents that target this ionic pump, like cardiac glycosides 272728Autosis is a variant of autophagic cell death, i.e., a form of RCD that isprecipitated by the molecular machinery for macroautophagy 11527Pyroptosis is a form of RCD that critically rely on the cleavage of gasdermin D(GSDMD) by inflammatory caspases, i.e., caspase-1 (CASP1), caspase-4 (CASP4),caspase-5 (CASP5) or caspase-11  1152930Morphologically, pyroptosis manifests with features that resemble (at least inpart) those of apoptosis 448990In summary, it remains to be formally demonstrated whether mitochondria are acore component of the signal transduction cascades that precipitate pyroptosisor whether they simply act as pyroptosis initiators in specificpathophysiological settings.Figure 1). Interestingly enough,evolutionarily ancient eukaryotes including Saccharomycescerevisiae mostly (if not exclusively) rely on mitochondrion-dependentforms of RCD 929596Caenorhabditis elegansDrosophilamelanogasterThe signal transduction cascades that precipitate RCD have become increasingly morecomplex with evolution, especially along with the acquisition of the eukaryoticstate and multicellularity 12131493In conclusion, mitochondria are quintessential for eukaryotic cells, not only as theymediate fundamental bioenergetic and anabolic functions, but also as they contributeto several  signal transduction cascades that precipitate RCD.


This longitudinal study aimed to investigate the prevalence of newly-started drinkers and their continuing drinking behaviors after the Great East Japan earthquake. Moreover, the relationships between newly-started drinking and psychological factor, disaster-related experience, and perceived radiation risk were examined. We used data from 37,687 pre-disaster non-drinkers who participated in the 2012 and 2013 surveys conducted in Fukushima. We defined newly-started drinkers as those who did not drink before the disaster but who began drinking after the disaster, based on information collected retrospectively. In 2012, 9.6% of non-drinkers began drinking, of which the prevalence of heavy drinkers was 18.4%. The prevalence of continued drinking among newly-started drinkers in 2013 was 53.8%. Logistic regression analyses revealed post-disaster newly-started drinking was significantly associated with being male, less than 65 years old, sleep dissatisfaction and psychological distress (Kessler 6 ≤ 13) when this model was adjusted for disaster-related experience and perceived radiation risk. Moreover, psychological distress and heavy drinking were significant risk factors for continued drinking among newly-started drinkers. Newly-started drinkers might use alcohol to cope with disaster-related stress. Thus, they may be targeted for disaster-related health services. Moreover, early intervention should encourage responsible drinking, since post-disaster heavy drinkers were likely to continue heavy drinking. The Great East Japan earthquake occurred on 11 March 2011, generating a massive tsunami and causing enormous damage to the Pacific Coast ,4. StudiSome disaster studies have reported that alcohol consumption increases following a disaster, due to psychological distress in affected individuals ,8. FurthAccording to a report following the Hanshin-Awaji earthquake in Japan, many victims showed increased symptoms of stress, difficulties caused by anxiety and sleep disturbance, and depressive symptoms or hopelessness . After tIn this study, we longitudinally investigated the prevalence of newly-started drinking and associated factors for two years after the Great East Japan earthquake. We also examined which factors are likely to pose a higher risk for continued drinking behavior among newly-started drinkers.After the devastating triple disaster in Fukushima Prefecture, the annual Fukushima Health Management Survey began in 2011 in order to monitor evacuees’ health and lifestyle conditions. This survey was designed to determine whether the triple disaster affected the evacuees’ physical, psychological, and socioeconomic wellbeing . We utilFor the current study, we used data from respondents in the 2012 and 2013 surveys who were at least 20 years old on 11 March 2011, and who reported in the 2012 survey that they were a non-drinker before the disaster. Among the 66,501 respondents in the 2012 survey, 37,687 of pre-disaster non-drinkers were analyzed in order to evaluate factors associated with post-disaster newly-started drinking .Among 180,604 residents in the target cohort, 66,501 responded to 2012 survey. After excluding the subjects missing gender, age and drinking behavior, 37,687 respondents of non-drinkers pre-disaster was analyzed. Of this group, 3569 evacuees began drinking post-disaster, including 2913 light drinkers and 656 heavy drinkers. Among these newly-started drinkers, 953 evacuees continued drinking, including 227 heavy drinkers, in the 2013 survey.First of all, we grasped non-drinkers in pre-disaster by self-reporting in closed questionnaire. Respondents in the 2012 survey self-reported their drinking habit in pre-disaster as “don’t drink or drinking only rarely (less than once per month),” and “current drinker (at least once per month).” Moreover, they answered their current alcohol consumption as “don’t drink or drinking only rarely (less than once per month),” “quit (history of drinking but not a current drinker),” and “current drinker (at least once per month) in the same survey conducted in 2012. Furthermore, we investigated the same questionnaire in the 2013 survey and grasped the respondents’ current drinking behavior .By these procedures, we defined newly-started drinkers and continuing drinkers after having begun. “Newly-started drinkers (having drinking behavior at least once per month) after this disaster” were as those who reported that they did not drink or drinking only rarely before the disaster and were a current drinker in the 2012 survey. Newly-started drinkers who reported being a current drinker in both the 2012 and 2013 surveys were labelled as “continuing drinkers (having drinking behavior at least once per month)”.Moreover, drinkers were further categorized into heavy drinking and light drinking groups, based on the amount of alcohol consumed on a typical drinking day. We defined heavy drinkers as those who consumed four drinks per day and more, and light drinkers as those who consumed four or less drinks per day. The reason was that the National Health Promotion of Japanese Ministry of Health, Labor and Welfare defined the heavy drinking as 6 Drinks per day in only on the days of drinking among both genders. However, this definition of heavy drinking might be a large amount for newly-started drinkers. Therefore, we utilized the cutoff point as “4 drinks per day” in only on the days of drinking, which is based on recommended limits of drinking in male (4 Drinks per day) in the National Health Promotion. By the way, 4-Drrink was defined as 120 mL of spirits , 480 mL of wine, 1000 mL of beer, or 360 mL of Japanese sake.The Mental Health and Lifestyle Survey mainly assesses lifestyle factors , disaster-related experiences, socioeconomic circumstances, general subjective health status, current mental health status, and perceived risks from radiation exposure . In ordeTo evaluate the relationship between health status and drinking behavior, general subjective health status and dissatisfaction with sleep were measured on a Likert scale . We defined those reporting ‘poor’ and ‘very poor’ general subjective health status as the ‘poor health’ group. Those who responded that their level of satisfaction with their sleep was ‘slightly dissatisfied’, ‘dissatisfied’, or ‘strongly dissatisfied’ were assigned to the ‘dissatisfied with sleep’ group.To assess mental health status among residents, we utilized the K6 scale to screen for non-specific psychological distress . Those sDisaster-related experiences, including experience of the tsunami and nuclear power plant accident, were utilized as adjustment factors. In this survey, the experience of nuclear power plant accident was categorized according to whether respondents heard an explosion or not. It was only natural that the experience of the earthquake would be an important disaster-related experience in this triple disaster; however, the vast majority of respondents to the Fukushima Health Management Survey have shared this experienced. For this reason, we excluded experience of the earthquake from our analyses.It has been reported that socioeconomic circumstances may affect evacuees’ psychological status . Thus, iRadiation exposure in the triple disaster was an unprecedented experience among the evacuees, and their perceived radiation exposure risk may have affected their disaster-related stress or psychological distress. We measured participants’ perceived risk of radiation exposure with the following question: “What do you think is the likelihood of damage to your health  in later life as a result of your current level of radiation exposure?” . This quFirstly, we excluded subjects who were missing age, gender, drinking behavior in pre-disaster and current before analyses . To assep < 0.05 level of significance. All statistical analyses were performed using IBM SPSS Statistics version 23.0 .Statistical significance was evaluated using two-sided, design-based tests with a The characteristics of respondents in the 2012 survey (including pre-disaster drinkers and non-drinkers) are shown in Among non-drinkers before the disaster, 3569 evacuees : 9.3–9.9%) reported that were current drinkers in the 2012 survey, of which 656  were heavy drinkers. Among these newly-started drinkers, 953 respondents  continued drinking at the time of the 2013 survey. The number of heavy drinkers was 227  in 2013 .Among the 2012 survey respondents, the prevalence of non-drinkers pre-disaster was 9.6% (95% CI: 9.3–9.9%). Of this group, heavy drinkers who have begun drinking post-disaster was 18.4% (95% CI: 17.1–19.7%), and light drinkers was 81.6% (95% CI: 80.4–82.9%). Among these newly-started drinkers, 53.8% (95% CI: 51.5–56.1%) of them continued drinking. Moreover, the prevalence of heavy and light drinkers in this group were 28.8% (95% CI: 21.1–26.5%), 72.2% (95% CI: 73.5–78.9%), respectively, in the 2013 survey.p < 0.01) and psychological distress  were significantly associated with newly-started drinking. General subjective health status was not a significant risk factor . Model 2 was adjusted by disaster-related experience, the effect of the disaster on socioeconomic status, and perception of radiation risks. Newly-stassrted drinking in 2012 was significantly associated with being male , aged under 40 years old , dissatisfaction with sleep , psychological distress , whose findings were not significantly different with the Model 1 results. General subjective “poor health” status was not a risk factor for newly-started drinking . In a comparison between heavy and light drinkers in Model 3, those who had been suffered severe psychological distress were more likely to drink heavily .We performed a multivariate logistic regression to examine factors related to post-disaster newly-started drinking in the 2012 survey . In Modep = 0.04) and heavy drinking behavior in 2012  were significantly associated with continued drinking in 2013, as with results in Model 1 , psychological distress  was significantly associated with continued drinking, whereas sleep dissatisfaction and disaster-related experiences were not significantly associated. Extended observation, therefore, could give different findings; those who have continued to drink while experiencing psychological distress might be more affected by the long-term changes in socioeconomic status or stressful lifestyle under evacuation ,21.However, there may be different factors associated with newly-started drinking among young people  compared to the middle-aged and elderly people who were affected by this accident. Therefore, we analyzed a subset of respondents who were over 40 years old. However, this did not show a significant effect in the 2012 and 2013 surveys shown in .In our findings, heavy drinking behavior (four drinks per day and more) among newly-started drinkers in 2012 was a significant risk factor for continued drinking behavior in 2013. Therefore, those who begin drinking post-disaster, especially those who drink heavily, should be high-priority subjects for disaster-related mental health services. By following up on their health condition and, if necessary, providing early intervention to encourage responsible drinking, it may be possible to prevent heavy drinking and continued drinking behavior ,18,19.Otherwise, disaster-related experience, disaster effects on economic status (loss of employment due to disaster) and perception of radiation risks did not have a stronger association with starting to drink behavior or continuing drinking behavior, compared with psychological distress. This was because these odds ratios of these variables were relatively small. The effects of the disaster on socioeconomic statuses, such as loss of employment, was hypothesized to be a risk factor for post-disaster newly-started drinking and continued drinking . It is tDue to the accident at the nuclear power plant, we considered that perception of radiation risk would have an impact on evacuees’ drinking behavior. According to a previous report regarding radiation risk perception among evacuees in Fukushima Prefecture, those who perceived a very high risk from radiation exposure tended to have psychological distress . Our finThe present study has several limitations. First, the response rate was less than 40% in the 2012 and 2013 survey. Therefore, the results of this study may not generalize to all evacuees within the areas specified by the government. Moreover, previous studies showed that the mental health status might have effects on response rate to survey, suggesting that non-response was associated with bad mental health status ,33. TherSecond, there was inadequate information on physical health status in the present study, such as the results of health checks. Although a history of the physical disease may affect drinking behaviors, the information on physical disease history could include some information bias because of self-reporting. Therefore, general subjective health status was utilized as a variable of physical health status even though it may be an insufficient variable. Finally, since there is a lack of information regarding the amount of alcohol consumed before the disaster, we could not examine change in the amount of alcohol consumed in the pre- and post-disaster periods. Furthermore, recall bias should be considered because we conducted these survey after this disaster by self-reporting, and collected pre-disaster drinking status retrospectively without validated measures such as AUDIT (the Alcohol Use Disorders Identification Test). This is an important limitation of this study.Despite these limitations, this study has multiple strengths. Studies regarding the association between alcohol consumption and disasters have been conducted using longitudinal analysis which determined the association between drinking behaviors and related factors. This was a large-scale survey of the evacuees who were residing within the designated evacuation areas in Fukushima; it can be utilized to assist evacuees in recovering from mental health damage. It is anticipated that psychological and socioeconomic issues related to newly-started drinking among evacuees will be taken into account in ongoing mental health service activities.Our findings showed that newly-started drinking was associated with being male, younger, dissatisfaction with sleep, and psychological distress following the disaster. Heavy drinking and psychological distress were risk factors for continued drinking among newly-started drinkers. It should be noted that newly-started drinkers who use alcohol to cope with disaster-related stress may be a suitable target group for disaster-related mental health services. Furthermore, it is essential to encourage responsible drinking and promote early intervention for heavy drinkers, since our findings show that newly-started drinkers who drink heavily are more likely than light drinkers to continue drinking. We hope our work will have implications for disaster-related mental health service providers.


In recent computerized cognitive training studies, video games have emerged as a promising tool that can benefit cognitive function and well-being. Whereas most video game training studies have used first-person shooter (FPS) action video games, subsequent studies found that older adults dislike this type of game and generally prefer casual video games (CVGs), which are a subtype of video games that are easy to learn and use simple rules and interfaces. Like other video games, CVGs are organized into genres  based on the rule-directed interaction with the game. Importantly, game genre not only influences the ease of interaction and cognitive abilities CVGs demand, but also affects whether older adults are willing to play any particular genre. To date, studies looking at how different CVG genres resonate with older adults are lacking.The aim of this study was to investigate how much older adults enjoy different CVG genres and how favorably their CVG characteristics are rated.A total of 16 healthy adults aged 65 years and above playtested 7 CVGs from 4 genres: casual action, puzzle, simulation, and strategy video games. Thereafter, they rated casual game preference and acceptance of casual game characteristics using 4 scales from the Core Elements of the Gaming Experience Questionnaire (CEGEQ). For this, participants rated how much they liked the game (enjoyment), understood the rules of the game (game-play), learned to manipulate the game (control), and make the game their own (ownership).Overall, enjoyment and acceptance of casual game characteristics was high and significantly above the midpoint of the rating scale for all CVG genres. Mixed model analyses revealed that ratings of enjoyment and casual game characteristics were significantly influenced by CVG genre. Participants’ mean enjoyment of casual puzzle games (mean 0.95 out of 1.00) was significantly higher than that for casual simulation games (mean 0.75 and 0.73). For casual game characteristics, casual puzzle and simulation games were given significantly higher game-play ratings than casual action games. Similarly, participants’ control ratings for casual puzzle games were significantly higher than that for casual action and simulation games. Finally, ownership was rated significantly higher for casual puzzle and strategy games than for casual action games.The findings of this study show that CVGs have characteristics that are suitable and enjoyable for older adults. In addition, genre was found to influence enjoyment and ratings of CVG characteristics, indicating that puzzle games are particularly easy to understand, learn, and play, and are enjoyable. Future studies should continue exploring the potential of CVG interventions for older adults in improving cognitive function, everyday functioning, and well-being. We see particular potential for CVGs in people suffering from cognitive impairment due to dementia or brain injury. Commercial video games are designed to be enjoyable, challenging, and capable of fostering sustained player engagement . Video gTo date, most video game training studies have used hardcore action video games  to demonstrate how video games can improve perceptual and cognitive abilities, notably those that are also subject to age-associated decline ,5. AlthoIn order to offer older adults and even patients more attractive forms of video game interventions, we suggest tapping the potential of CVGs as an enjoyable activity to improve cognitive functions and emotional well-being. By better understanding the types of CVGs older adults enjoy playing and the specific game characteristics they find appealing, we hope to identify CVG genres and game characteristics that might raise the motivation of participants in future video game interventions.What makes CVGs an excellent choice for an older adult population is their promise to “eliminate any possible barrier to someone enjoying the game” . Unlike To reach this goal, CVGs follow four casual game design values: “Acceptability” refers to the appeal for a wider, heterogeneous group of players. To this end, CVGs borrow themes familiar to the social context of the player that are nonviolent and foster positive emotions and growth. “Accessibility” makes sure that players with different cognitive and physical skill levels can quickly learn to play the game. “Simplicity” aims to lower the cognitive load on the player through simple and minimized game elements as well as easy rules and goals. Finally, “flexibility” assures that CVGs adapt to players and integrate into their everyday life. For this, CVGs are designed to be error-forgiving, adapt the difficulty level to the player, and can be easily stopped and replayed ,13,14.Interestingly, CVGs further try to provide players with positive outcomes outside of the game such as mental exercise, relaxation, social and playful activity . This toThe notion of video game genre that allows organizing games into categories is crucial in connection with CVGs for three reasons: Whereas it is agreed that CVGs, as a whole, reduce usability barriers and are the most enjoyed and motivating type of video games, research into preferences for CVG subgenres among older adults is lacking . This isAlthough several recent studies have addressed usability barriers and used survey methods to assess game preferences in older adults ,16,23,24In this study, we wanted to identify CVG genres that older adults enjoy and are willing to play most, and how favorably their CVG design values are rated. Given the explorative nature of this study, the following research questions rather than hypotheses were formulated. Do the CVGs provide enjoyment and is there a preference for specific CVG genres? Do the CVGs meet the casual game design values  and are they suitable for older adults?In total, 16 healthy older adults  aged between 65 and 84 years  participated in this study. Participants were recruited from the Seniors University of Bern, Switzerland. All participants had normal or corrected-to-normal vision. Exclusion criteria were a diagnosis of dementia or mild cognitive impairment and fine motor skill impairments leading to inability to handle a tablet computer. All participants provided signed informed consent in accordance with the latest version of the Declaration of Helsinki and were rewarded with two cinema tickets for their participation. The cantonal ethics committee of Bern, Switzerland  granted the ethics approval.For this study, we selected 7 single-player, tablet-based CVGs  availablFor genre-specific comparisons, we selected casual games from 4 casual game genres , following video game genre classifications based on both the cognitive skills they involve  and the For the casual action games  after completion of each game . The CEGFor this study, we selected 5 scales of the CEGE . Casual game genre preference was measured using the enjoyment scale (3 items) reflecting the extent to which each game was enjoyed and encouraged replayability. Three scales were used to examine to what extent the selected casual games met the casual game values ,13,34 asThis study used playtesting sessions that combine questionnaires with the opportunity of hands-on playing of video games to quantify the player’s attitudes, opinions, and perception of different CVG genres. This approach is well suited for persons with no previous game experience and also far more informative than mere interviews and surveys in active gamers . For theThe participant’s evaluation of the game experience with the casual games was assessed with 39 closed-response questions that participants answered using a closed 5-point Likert-type scale. The 10 negatively worded items were reverse-coded by subtracting the value from 6. Scores for each of the 10 scales of the CEGEQ were then calculated by averaging the item ratings corresponding to each scale and dividing them by 5. We removed item 24 (“I felt guilty for the actions in the game”) from the CEGEQ as we specifically selected nonviolent video games.First, separate repeated measures one-way Analyses of Variance (rmANOVA) with casual game genre as a within-subject factor were performed by using SPSS for Windows  to test whether the video game genre had a significant effect on each of the 5 CEGEQ scales of interest . An alpha value of .05 was used to determine significance.F test, whereas all other fixed-effect parameters were tested using a conditional t test. Pairwise comparisons of the estimated marginal means were performed between the different video game genres using Bonferroni-Holm correction for multiple testing.Second, linear mixed effect models (LMEM), fit by restricted maximum likelihood estimation (REML) were performed  and the package lme4 ) for eacAll 16 participants reported having access to and using desktop or laptop computers on a daily or weekly basis, whereas only 5  participants  reported having ever played, and that they are currently playing computer video games. All 5 participants reported playing games preinstalled on Desktop PC computers  on a daily to monthly basis. None of the participants reported having ever played video games on tablet-computers.The results of this study indicate that the mean participant’s ratings of the casual games were above average  across all CEGE scales considered in the analysis. The order of casual game enjoyment or genre preference from the most enjoyed to the least enjoyed was as follows , total: F1, 2.977=4.794, P=.005, η ²=.220); game-play ; control ; and ownership  ratings. There was, however, no significant difference in environment ratings  across CVG genres. To adjust the effects of video game genre for the potentially confounding effect of the graphics and audio of the video games (“environment”), the effect of video game genre and environment were analyzed within a LMEM for each CEGE scale. In addition, the effects of personal background variables  were included in the LMEMs (P<.001) and a trend for positive effects of environment ratings (P=.05). For game-play ratings, there was a significant global effect of video game genre (P<.001) and a significant negative effect of game experience (P=.01). For control ratings, there was only a significant global effect of video game genre (P<.001), whereas there was a significant global effect of video game genre (P=.01) and a significant positive effect of environment ratings (P=.03) on ownership ratings. The linear mixed-effects model was further used to perform all 21 pairwise comparisons between the 7 CVGs for each CEGE scale showing significant global effects of the factor video game genre. Pairwise comparison of the estimated marginal means for enjoyment indicate that the casual puzzle video game (Flow Free) was significantly more enjoyed than casual simulation racing  and sports  video game. Game-play ratings of casual action no shooting video game scored significantly lower than those for the casual puzzle video game , and the casual simulation racing  and sports  video game. Pairwise comparisons further indicated that control ratings for the casual puzzle video game (Flow Free) were significantly better than those for both the casual action no shooting  and shooting  video game as well as for the casual simulation racing  and sports  video game. Furthermore, the second casual puzzle video game (Bejeweled) was rated better in terms of control than the casual simulation racing video game . Finally, pairwise comparisons on the effect of video game genre on ownership revealed that both the casual puzzle video game  and casual strategy video game  were rated better than the casual action no shooting video game.As hypothesized, there was a significant main effect for CVG genre on enjoyment  over the casual simulation games . Nevertheless, all CVGs were given good enjoyment and replayability ratings. This finding is strongly in line with findings from previous survey studies in active older gamers ,24,37 anHowever, we did not find evidence to support a general dislike for the action game genre (no shooting and shooting) reported in other studies , as thisAs for the third most liked casual strategy game genre (Plants vs Zombies), we were not able to relate this finding to survey and other playtest study findings; as, to the best of our knowledge, there have been no studies on enjoyment and usability using strategy games in older adults. However, strategy games have successfully been used in 1 video game training study with older adults . FinallyTaken together, these findings indicate a positive overall attitude of older adults toward tablet-based casual games, and that they are very open and willing to try out new technology when given an opportunity to play. Of note, environment  ratings remained a contributor to enjoyment when controlling for all other effects. Whereas environment ratings did not differ across genres, the visual presentation of a CVG seems to contribute to overall enjoyment of CVGs.We also find hands-on evidence to support the notion that casual games provide a set of game characteristics that are suitable for a senior audience in terms of usability . OverallThe gameplay ratings, that is, how participants judged the rules and underlying story (scenario) of the games were satisfactory, with the casual simulation and puzzle video games being easier to understand than the casual action (no shooting game) video game. The fact that the least liked games were rated easiest to understand is hardly surprising, as simulation games make use of real-world concepts and rules already known to the player and thus, at least in terms of rules of the game, are particularly suitable for an older audience with little or no game experience . In termThe latter two findings reflect the above-mentioned notion that action games are not as much appreciated due to the speed of movement and intense interaction, which is reflected in the lower control and ownership ratings . These fOf note, the least enjoyed casual simulation (sports and racing) video games differed inherently from the others in how players had to control the game. Other than using single tapping or sliding movements as game input, these games were unique in that they required players to perform tilting (to steer the car in the racing game) or continuous drag movements (to move the ping-pong paddle in the sports game). This additional load on the interaction with the game should be considered when comparing ratings of enjoyment and interaction with the game.This study is not without known methodological limitations regarding problems in obtaining a representative sample of older adult players . Of noteAlthough special attention was paid to gender-inclusivity during the selection of the casual games, we did not include gender as a factor in the analysis of genre preference and interaction with the game, owing to the smaller number of female participants.In addition, playing time for each video game was limited to around 5 min, allowing us to collect first impressions and ease of interaction, rather than long-term experiences with the video games. It therefore remains unclear whether the CVG genre preferences persist over extended experience with the game .The goal of this study was to find out how well different CVG genres met casual game characteristics and how this led to game enjoyment in a population of older adults. We argue that our findings can be matched with a recent position De Schutter and Abeele  take in To address this issue, the manifesto states that video games should primarily provide older adults with a meaningful and playful activity. We were able to confirm this with the general finding that casual games were well-enjoyed and participants were willing to play them. This is closely related to their second claim that video games should focus on challenge and personal growth and rather than simply combatting age-related cognitive decline. Although we did not address this, we believe that this claim is easily met by the positive themes CVGs are characterized by and that these games use difficulty adjustments to optimally challenge the player. Finally, in their third claim the authors propose that video games should offer a diversity to accommodate the heterogeneity of older adults in their cognitive and physical skill levels, backgrounds, and preferences. Again, it is our understanding that it is in the very nature of CVGs to satisfy this demand.In line with recent findings, this study confirms a special preference of older adults for the puzzle game genre. It would be interesting for future studies to capitalize on this in future video game interventions for older adults. It also appears worthwhile to conduct video game interventions using multiple game genres, as only few studies have looked at the training benefits of different game genres in older adults . We ther


Solid–solid transitions between crystals follow diffusive nucleation, or various diffusionless transitions, but these kinetics are difficult to predict and observe. Here we observed the rich kinetics of transitions from square lattices to triangular lattices in tunable colloidal thin films with single-particle dynamics by video microscopy. Applying a small pressure gradient in defect-free regions or near dislocations markedly transform the diffusive nucleation with an intermediate-stage liquid into a martensitic generation and oscillation of dislocation pairs followed by a diffusive nucleus growth. This transformation is neither purely diffusive nor purely martensitic as conventionally assumed but a combination thereof, and thus presents new challenges to both theory and the empirical criterion of martensitic transformations. We studied how pressure, density, grain boundary, triple junction and interface coherency affect the nucleus growth, shape and kinetic pathways. These novel microscopic kinetics cast new light on control solid–solid transitions and microstructural evolutions in polycrystals. et al. show a transformation pathway that starts off as being martensitic then switches to diffusive at the single particle level in a colloidal system under pressure.Solid-solid transitions between different crystalline structures have broad implications in earth science, steel and ceramic materials. Peng  Solid–solid (s–s) transitions are arguably the most common type of structural phase transitions, and prevail in natural and man-made systems1568Reconstructive s–s transitions are theoretically difficult because an order parameter cannot be defined easily without a group–subgroup relation in the lattice symmetries217111517Colloids are outstanding model systems for phase transition studies because micron-sized colloidal particles can be imaged directly by optical microscopy and their thermal motions can be tracked by image processing2225262728293026272830Here we study the reconstructive s–s transition between square and triangular lattices, which is one of the simplest s–s transitions without a group–subgroup relationN-isopropylacrylamide) (NIPA) microgel spheres whose diameter, σ, linearly changes from 0.76 μm at 26.4 °C to 0.67 μm at 30.6 °C Δ transition along a tilted path in the phase diagram . The s–s transition can occur when Tamb<Ts−s<Tamb+δT<Tm , where Tm and Ts−s correspond to the melting and s–s transitions, respectively. The heating effect is uniform enough in the central π(38 μm)2 area of the focal plane  of the crystal, which lasted for approximately an hour and slowly varied over several minutes Δ transitions for different n. When nuclei are larger than H, their shapes were roughly uniform in the z direction, and so we mainly monitored the surface layer. We record particle motions using a charge-coupled device camera at 10 frames per s. Particle positions were tracked from the image analysisIn most experiments, the colloid was partially filled in the sample cell with air trapped in the corners. Gently pressing one corner induced a tiny drift (flow rate  minutes . The cryn✓→liquid nucleus→(n−1)Δ, which was confirmed in simulationPreviously we reported the s–s transition in such colloidal thin films under isotropic stressν≳10 nm s−1 (42ν<10 nm s−1), we previously found that the precursors were in fact particle-swapping loops rather than the conventionally assumed crystal defectsν>10 nm s−1 were oscillating dislocation pairs, which represent a new type of nucleation precursor. Such martensitic type of precursor has not been suggested before. Here we call it martensitic precursor, which is beyond the conventional structural description , and thus have more space to rearrange themselves and transform into a Δ-lattice. Consequently, facet VI propagated faster than facet III in 1L near facet I was higher than that near facet IV, thus facet I grew faster  due to the early-stage martensitic precursor  as shown in φ and high ν, the early-stage nucleation was similar to that in φ  and high ν  but a shorter incubation time than does Most crystals contain numerous grain boundaries and triple junctions, hence their effects on the s–s transition are particularly important. We found that the nucleation at grain boundaries and triple junctions can exhibit different kinetics. When  nucleus . The earAs the nucleus grew larger, it developed two coherent facets for a lower interfacial energy. The other two incoherent facets oriented 45° away from the square lattice. Such a 45° inclination angle  can be sν≲10 nm s−1. The mismatch angles between the neighbouring lattices were all 60°. An equilateral triangle shape could make all nucleus' facets coherent for a lower energy. Since the interfacial energies γcoherent<γliquid<γincoherent in this colloidal systemL mainly occurred at the junction of the nucleus and grain boundaries rather than on the nucleus facets because the facets were coherent and tended to be flat. Particles were attached to the coherent facets from the junctions of grain boundaries and formed high-energy kinks, which induced nucleus growth, similar to those in We found that applying a small flow is not the only way to suppress the intermediate liquid nucleus during the ✓−Δ transformation. n✓→(n−1)Δ transitions follow the nucleation process, which is a signature of the first-order transition. The rich behaviours of different nucleation pathways are summarized in All homogeneous and heterogeneous n✓→n−Δ transitφ for colloids), that is, incomplete martensitic nucleation  so that the melting volume fraction of the 3D crystal was the same as that of hard spheres  resulted in either n✓→(n−1)Δ→liquid, n✓→liquid or nΔ→liquid transitions, but not nΔ→n✓→liquid or n✓→nΔ transitions. These behaviours were confirmed in our experiments. Here we only focused on the temperature regime near the n✓→(n−1)Δ transition, which occurs below the (n−1)Δ→liquid transition point. For 5✓→4Δ transitions, H/σ≃3.9 and 4.1, and φ≃0.62 and 0.55, before and after the local heating, respectively. The samples were prepared as in our earlier thin-film melting experiments58H was roughly determined by the added volume of colloidal suspension and its spreading area. When the colloidal suspension had spread over approximately half of the 18 × 18 mm2 coverslip area, we hermetically sealed the sample with epoxy glue and fixed its thickness. The air trapped at the corners and edges is more sensitive to pressure changes and can induce a small flow in the colloid when a mechanical force is applied. We manually used an objective attached with a spring to press the coverslip and apply the pressure. The measured flow rates in the fields of view were very small, and thus their fluctuations were relatively large  microgel spheres were synthesized and dispersed in an aqueous buffer solution with 1 mM acetic acid. These microgel spheres were slightly charged with short-range steric repulsions . The effspension 53. Since.4% ref. . Therefoly large . Neverthly large , 2, 3. Aly large , and alsn✓–(n−1)Δ interfaces  can quickly stabilize in 3 s after the light was turned on. Therefore, the observed nucleation processes were in steady states.The typical grain size of the polycrystals ranged from 10 to 300 μm. A 0.4 μl colloidal suspension usually assembled into four layers at the centre and five or six layers near the edges. Therefore, the wall bending was small and the thickness was rather uniform over a field of view of ∼100 μm. The glass surfaces were rigorously cleaned so that particles would not stick to the walls. The refractive indexes of NIPA spheres and water are very close because more than 90% of the microgel was water. Consequently, bulk layers can be seen clearly in images even under the bright field when the spheres were packed into a crystalon point . This opL to characterize the volume fraction φ. The results can be directly compared with L in atomic crystals. To avoid the slow divergence of mean-square displacement (MSD) in 2D, a modified dynamic Lindemann parameter L based on the local coordinates of neighbouring particles is often usedSince the diameter and volume fraction cannot be determined exactly for soft spheres, we use the Lindemann parameter a is the lattice constant measured from the position of the first peak of the radial distribution function g(r), Δrrel is the relative neighbour–neighbour displacement, Δui is the displacement of particle i, particles i and j are nearest neighbours, and rrel(t))2〉 describes the displacement relative to a previous time. L is measured from −2 to +2 s around each frame and is colour-coded in L. A dislocation can enhance the L value of its four layers of neighboursm=4 and 6 correspond to four- and six-fold symmetries, respectively. θj is the orientational angle of the bond between particle i and its nearest neighbour j. nni is the number of nearest neighbours for particle i. The nearest neighbours are identified from the Delaunay triangulations, which yield 〈nn〉=6 for any distribution of particles in 2D. Hence it is ideal for triangular lattices, but not for square lattices whose 〈nn〉=4. Therefore, the nearest neighbours for square lattices are further confined to those particles within a distance of <1.2a, which is the midpoint between the lattice constant a and the distance to the second nearest neighbour where urs ref. . . m=4 aur  ref. . A crystf.  ref. . A ✓- orThe data that support the findings of this study are available on request from the first author Y.P. or the corresponding author Y.H.How to cite this article: Peng, Y. et al. Diffusive and martensitic nucleation kinetics in solid-solid transitions of colloidal crystals. Nat. Commun.8, 14978 doi: 10.1038/ncomms14978 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures, Supplementary Notes and Supplementary ReferencesThe precursor, nucleation and growth processes in a locally defect-free region shown in Figure 1. The left part is the raw video and the right part is the same video but with the colour-coded Lindemann parameters. 40 × real time.The formation and motion of dislocation pairs and the martensitic nucleation shown in Figs. 1a-l. Real time.The precursor, nucleation and growth processes near a dislocation shown in Figs. 2a-g. The left part is the raw video and the right part is the same video but with the colour-coded Lindemann parameters. 40 × real time.The formation and motion of dislocation pairs and the martensitic nucleation shown in Figs. 2a-d. Real time.The growth of a nucleus on a grain boundary. The upper facet is incoherent whereas the lower one is coherent. The left part is the raw video and the right part is the same video but with the colour-coded Lindemann parameters. 40 × real time.The incomplete nucleation process shown in Fig. 4. The left part is the raw video and the right part is the same video but with the colour-coded Lindemann parameters. 40 × real time.2 (right) in during the nucleation shown in Fig. 1 and 7. The colour-coded γ and D2 are same as those in Figs. 6 and 7. 5 × real time.The strain fields of γ (left) and DThe nucleation and growth processes near a grain boundary shown in Fig. 8. The left part is the raw video and the right part is the same video but with the colour-coded Lindemann parameters. 40 × real time.-1) shown in Fig. 9. The left part is the raw video and the right part is the same video but with the colour-coded Lindemann parameters. 40 × real time.The nucleation and growth processes near a triple junction with a negligible flow (2 nm s


To determine the prevalence and factors associated with malnutrition among infants with Cleft palate and/or cleft lip (CP+/-L) at Comprehensive Rehabilitation for Uganda Hospital (CoRSU) in Uganda.This was a cross-sectional study done on infants with CP+/-L and their caretakers admitted between November 2013 and October 2014 at CoRSU hospital which was the study setting. A questionnaire was answered by the infants’ caretakers. The main outcome measure, malnutrition was defined and classified based on Z-scores obtained using the W.H.O Z-calculator in which weights of the infants in kilograms and lengths in centimeters respectively were placed. The values obtained were expressed as a proportion using all enrolled infants with CP+/-L as denominator. Multivariable analysis was used to determine the risk factors.A total of 44 infants with CP+/-L were enrolled. Of these, 77% were below 4 months of age and 97.7% were immunized. The male-to-female ratio was 1.06:1. About 59% had unilateral CP+/-L. A total of 93.2% were delivered at term with 69.4% having a birth weight greater than 3 kg. Generally, 68% were malnourished, with the highest burden among females (71.4%), infants below 4 months (73.5%) and those with unilateral CP+/-L (77%). About 57% had moderate-to-severe malnutrition. There was delayed supplementation to breast milk, with cow-milk as the main supplemental feed for all the infants. In the multivariable analysis, factors associated with malnutrition included, having caretakers lacking nutritional information post-delivery , low birth weight , and having less than 10 feeds/day .CP+/-L infants born in Uganda suffer a high-burden of malnutrition. Preventional strategies are needed with focus on proper feeding information. Research on cost-effective feeds, feeding methods and reasons behind gender disparities in these infants is also required. Cleft palate and/or cleft lip (CP+/-L) are among the most common congenital anomalies, with an incidence of 1/1000 live births globally . ChildreMalnutrition in these children with CP+/-L is majorly associated with feeding difficulties including the failure to generate sufficient suction pressure during feeding hence affecting the attachment to the breast/artificial nipple, milk extraction, bolus organization and retention of the bolus before swallow initiation . MalnutrDespite the availability of surgical corrective interventions, few CP+/-L children are returned for their cleft palate surgical operation  in our setting a phenomenon hypothesized to be due to a likelihood of death before scheduled date of operation potentially due to consequences of malnutrition . Good nuHowever, adequate information on the prevalence and factors associated with malnutrition in CP+/-L infants is still lacking in our setting.In this study we aimed at establishing the prevalence and associated factors of malnutrition among infants with CP+/-L at CoRSU hospital.Determine the prevalence and associated factors of malnutrition among infants with CP+/-L at CoRSU Hospital.Across sectional study was carried out at Comprehensive Rehabilitation Services of Uganda (CoRSU) Hospital, a specialized plastic and reconstructive, as well as orthopedic surgery private facility. It is located along Entebbe road. They offer free cleft surgery and receive patients not only from Uganda but from other countries including South Sudan.A total of 44 admitted infants with CP+/-L between November 2013 and October 2014were consecutively enrolled in the study after obtaining consent from the primary caretakers. There were no primary caretakers that declined to participate or withdraw from the study during the study period.A pre-tested semi-structured questionnaire was developed and administered by trained research nurses to all caretakers of infants with CP+/-L. The questionnaire included caretaker and infant’s baseline characteristics, and infant’s nutritional characteristics. The post delivery nutritional information sought from the caregiver included, whether the caregiver had been advised on any techniques, positions and options available for feeding their infant. Information that was also sought regarding the feeding of the infants included the type of feed and how often it was given. Infant’s clinical examination including type of cleft palate and anthropometry measurements was done by an experienced doctor. Anthropometry included weights and lengths done to the nearest 0.01 kg and 0.1 cm respectively using a digital weighing scale and a length board. Infants who were found to have low weights for length after comparing with the N.C.H.S reference values as required by W.H.O were then sent to the nutrition unit at the hospital as per the protocol. Double-data entry and validation were done using EpiData software .Means and proportions were used to describe the socio-demographic characteristics of the infants with CP+/-L and their caregiver. Nutritional characteristics were stratified using a cut off of four months which was chosen arbitrarily. Weight-for-length Z-scores were calculated using a W.H.O Z-calculator. The prevalence and severity of malnutrition was expressed as percentages with corresponding 95% Confidence Intervals. Classification of malnutrition based on severity followed the standard as recommended by WHO.Chi-square and logistic regression was carried out for analysis of associations. All variables with p-value ≤ 0.2 were included in multivariable analysis. A significance level of 5% was considered. Unadjusted and adjusted Odds ratios with corresponding 95% CI were reported.The caretakers were predominantly mothers (98%), aged less than 30 years (66%), HIV negative (79.6%), and had delivered from a Health facility (68.2%). Majority had acquired some post-delivery nutritional information from a health facility 81.5%). About half had a primary level of education (54.6%). The infants were predominantly below 4 months of age (~77%) and immunized (97.7%). The infants’ male-to-female ratio was 1.06:1. Most infants had unilateral CP+/-L (59.1%), were delivered at term (93.2%) and had a birth weight of more than 3 kg (69.4%). See Table .5%. AbouA total of 17 (50%) versus 4 (40%) infants below and above 4 months respectively were solely breastfeeding. Complementary feeding was reported in 17 versus 4 infants below and above 4 months respectively. Cow’s milk was the most commonly used complementary feed. About half of the mothers reported inability of the baby to suckle as the main reason for not breast feeding. See Table About 30 infants  were malnourished, with the highest burden among females (71.4%), infants below 4 months (73.5%) and infants with unilateral CP+/-L (77%). Generally, 57% had moderate-to-severe malnutrition  as compared to their counterparts. Also, infants who had less than 10 feeds per day had 4.9 times higher odds of having malnutrition  as compared to those with above 10 feeds/day. Infants born with a low birth weight had 3.4 times higher odds of having malnutrition  method proposed by the W.H.O. was not used to define malnutrition. In the study done in Brazil  the prefSome of the studies such as the one conducted by Mehkarkar and Chaudhari  defined The high malnutrition seen in the infants less than 4 months was probably due to the infants’ inability to breastfeed adequately. This was also noted in the study done by Cubit et al . ExclusiThe prevalence of malnutrition among females was higher than that of males and the overall prevalence. The cause of this imbalance needs further investigation as very few studies evaluating the problem have focused on gender differences. Literature from elsewhere seem to point to a gender imbalance as regards to children’s nutrition and access to health care. A study done in Pakistan showed that male children were favored more in the allocation of health care compared to the females . Could tMalnutrition was also found to be higher among the unilateral CP+/-L infants compared to their bilateral counterparts. Could the severe facial disfigurement and apparent difficulty in breast feeding in bilateral CP+/-L have forced the mothers to seek medical attention earlier than their unilateral counterparts? This could have led to the differences observed.Regarding the associated factors, the lack of provision of nutritional information was associated with malnutrition among our infants with CP+/-L. Provision of nutritional information has been shown to be associated with improved outcomes. In the study performed in the United Kingdom , they noOur study noted that children who had fewer than 10 feeds a day were more likely to be malnourished. This could have been due to failure of the infant to breastfeed. The most common reason for not breast feeding was failure of the infants to suckle Table  which haThe Study showed that the risk of malnutrition was higher for CP+/-L infants born with a low birth weight. This finding is similar to studies done in Oman  and MalaThe combination of lack of provision of nutritional information following the delivery of a CP+/-L infant, number of feeds less than 10 received per day by the CP+/-L infant and low birth weight was found to be the greatest predictor of malnutrition among CP+/-L infants.This is the first study in our setting to explore the prevalence and severity of malnutrition with its associated factors among infants with CP+/-L.This work described malnutrition among infants with CP+/-L at a specialized plastic surgery hospital in Uganda.Like other studies previously reported, there was a high prevalence of malnutrition in this population. The low study participants obtained for the study was a major study limitation. However, the Low recruitment of study subjects concurred with studies done in a similar setting that showed few infants with CP+/-L were being seen at the time of their operation probably because they were dying earlier . MalnutrWe managed to describe the severity of malnutrition according to the WHO classification for malnutrition which no other studies had done in our setting. The fact that most children had severe malnutrition further strengthened the need for aggressive nutritional management for infants with CP+/-L with the help of the motivated caretaker.Many of the factors associated with CP+/-L were directly related to the caregiver status. There is therefore need for integration of everyday healthcare practices of the caregivers into CP+/-L surgical services to reduce the risk of malnutrition among these infants.Finally, longitudinal and interventional studies will be needed to better determine causation in many of the factors noted and to improve outcomes so that we prevent malnutrition in infants with CP+/-L.Proper Nutritional information should be readily available for all mothers who give birth to infants with CP+/-L.The nutritional information available should include methods of assisted feeding, feeding techniques such as positioning and follow up. Further studies may be required to assess the different modalities for assisted feeding in order to come up with the most applicable method for these caretakers to adopt in our setting.There is need to increase public awareness of the likely hood of malnutrition among infants born with a cleft palate. This would help emphasize nutrition both for the caretakers and care providers of these infants.Further studies are required to elucidate the role of culture in access of health care as regards the gender of the infants and perceptions of the cleft conditions.There is need to strengthen the link between general health care and maternal health care in our setting. We need to re-emphasize for example the need for proper maternal nutrition, the avoidance of smoking in pregnancy among others.