The selectivity for a specific human Carbonic Anhydrase (hCA) isoform is an important property a hCA inhibitor (CAI) should be endowed with, in order to constitute a valuable therapeutic tool for the treatment of a desired pathology. In this context, we developed a chemoinformatic platform that allows the analysis of the structure and selectivity profile of known CAIs reported in literature, with the aim of identifying structural motifs connected to ligand selectivity, thus providing useful guidelines for the design of novel ligands selective for the desired hCA isoform. The platform is able to perform ultrafast structure and selectivity analyses through ligand fingerprint similarity, with no need of structural information about the target receptor and ligands’ binding mode. It is easily accessible to the non-expert user through the implementation of a KNIME Analytic Platform workflow and could be extended to analyze the selectivity profile of known ligands of different target proteins. At present, seven different genetic families of CAs have been widely acknowledged: α-, β-, γ-, δ-, ζ-, η- and θ-CAs. All these enzymes catalyze the zinc-mediated reversible hydration of CO2 in HCO3– and H+, although some secondary catalytic reactions have been discovered and reported for at least some classes of CAs,,Human Carbonic Anhydrases (CAs) constitute a superfamily of metalloenzymes characterized by the presence of a zinc  associated with their AID and selecting only ligands whose Ki values were determined in the laboratory of Prof. Claudiu T. Supuran in order to minimize the biases associated to the application of different experimental procedures. Subsequently, for those ligands presenting more than one Ki value  for the corresponding hCA, the different activity values were averaged prior removal of all potential outliers, thus obtaining a reliable mean Ki value. Finally, the structures of all ligands in SMILES notation were automatically downloaded from PubChem database. The thirteen refined datasets of ligands were then merged into a single spreadsheet containing the records of all compounds presenting a Ki value for at least one out of the 13 hCA isoforms considered in this study. Each ligand record included the CID code and the SMILES string of the compound, together with at least a Ki value. The full list of CAIs was then imported into Instant JChemAll data related to known hCA inhibitors were retrieved from PubChem databaseKi values for both hCA II and IX, corresponding to 1756 compounds. The software was also used to calculate the selectivity index (SI) of each ligand, defined as the ratio between hCA II and hCA IX Ki values. Based on this definition, a ligand with a 10-fold selectivity for hCA IX over hCA II would show a SI = 10. Analogously, Instant JChem was used to select the 1388 ligands with Ki values for both hCA II and XII and to calculate their SI values  to be used for the hCA XII/hCA II selectivity profile analysis, as well as to select the 1322 compounds with Ki values for hCA II, IX and XII employed in the double selectivity analysis.For the hCA IX/hCA II selectivity profile analysis, Instant JChem was used to select a subset of ligands showing The generation of ligand fingerprints and the calculation of the Tanimoto similarity index were performed using the pybel module of OpenBabel softwareon, ligands with SI > 5 and thus selective for the on-target (hCA IX or hCA XII); (b) Loff, ligands with SI < 0.2 and thus selective for the off-target (hCA II); (c) Lns, ligands with 0.2 < SI < 5 and thus non-selective. Based on this classification, the selectivity score (Sscore) of each cluster was calculated as follows:The clustering analysis was performed using the software orange-canvasscore values would predominantly include ligands with selectivity for the on-target, while negative Sscore values indicate clusters mainly populated by compounds with selectivity for the off-target. In the double selectivity analysis, two different Sscore values relative to hCA II/IX and hCA II/XII selectivity were calculated for each cluster of ligands.Therefore, clusters with positive SFile Reader node was used to import the full list of hCA ligands previously generated, while two String Input nodes were employed to select the on-target and the off-target to be considered in the analysis. Within the meta node, two initial Row Filter nodes were used to select from the full list of hCA ligands only those showing Ki values for the desired hCA isoforms, thus obtaining the proper dataset of compounds to be analyzed. An RDKit Fingerprint node was used to generate MACCS fingerprints for the dataset ligands, which were employed for the calculation of the ligand similarity matrices. Ti and Td values for all possible fingerprint pairs were calculated using a Distance Matrix node applying the same formula reported above, while the hierarchical clustering analysis was performed through two different KNIME nodes: a first one generating the clusters and a second one assigning the cluster labels to the different dataset ligands. The same clustering algorithm and parameters employed in orange-canvas were also used in KNIME. The Sscore values relative to the generated clusters were obtained through a KNIME node encoding our in-house python scripts that calculate the SI value associated to each ligand and the Score associated to each cluster, based on the above reported equation. A further Row Filter node was then used to select the clusters with Sscore > 70. The final nodes added at the end of the workflow produced two different visual outputs: (a) a Render to Image node and an Excel Writer node were used to generate a spreadsheet including 2D structures, CID codes and SI values of the ligands included in clusters with selectivity for the on-target (Sscore > 70), together with the number of their corresponding cluster; (b) a Marvin View node was also employed to produce a compound table showing the ligands grouped within the selective clusters as visualized in Instant JChem software, which included the 2D structures of the ligands and the same properties reported in the spreadsheet.A KNIME workflowKi value were retained in the refined datasets, while ligands associated with activity values expressed through different metrics were filtered out . The 13 final datasets of CAIs were eventually merged into a single database to be used for selectivity profile analyses, including all compounds presenting a Ki value for at least one out of the 13 hCA isoforms considered in this study. However, since the majority of CAIs are characterized by the presence of a sulfonamide or sulfonamide-like moiety acting as zinc-binding group (ZBG), and the most potent CAIs belong to this class of compoundsKi values for one or more hCA isoforms. As a first step in the development of our selectivity analysis platform, we focused on gathering a large amount of bioactivity data related to hCAs inhibition and the corresponding structures of small-molecule ligands experimentally tested for hCAs inhibitory activity. For this purpose, we searched the PubChem database and retrieved the SMILES strings of all compounds for which the result of a biological assay on at least a single hCA isoform was stored. In this way, we created 13 initial targeted datasets of ligands, each including their structure and bioactivity information related to a specific enzyme out of the 13 hCA isoforms that were considered in our analysis: hCA I–IV, Va, Vb, VI, VII, IX and XI–XIV. These preliminary data sets were then refined in order to obtain ensembles of compounds with bioactivity data that could be safely compared to each other, thus limiting the biases associated to experimental procedures. For this reason, only compounds whose inhibitory activity for the corresponding hCA isoform was expressed by a Ki values for both hCA IX and hCA II, and we calculated their selectivity index (SI), defined as the ratio between their Ki values for hCA II and hCA IX. Subsequently, the structures of these ligands were translated into fingerprints, which were then compared to one another by calculating the Tanimoto similarity index (Ti). In this way, it was possible to perform a rapid evaluation of the reciprocal structural similarity among all 1756 ligands belonging to this subset . Such analysis was performed four times, using four different fingerprint types: FP2, FP3, FP4 and MACCS. Each of these fingerprints encoded the structures of the dataset ligands in a different way and thus different similarities and corresponding Ti values were obtained for the same ligand pairs. For all possible pairs of ligands included in the dataset, the Ti values of the corresponding fingerprints were calculated, together with the selectivity ratio (SR), defined as the ratio between the SI of two ligands expressed in logarithmic scale. In this way, we could evaluate the correlation between ligand similarity in terms of structure (described by the Ti) and isoform selectivity (described by the SR). A pair of ligands with a similar selectivity profile  will show an SR close to zero, while pairs of ligands with different selectivity profiles  will show an SR far from zero. The results of each similarity analysis were evaluated by plotting Ti versus SR values calculated for each ligand pair within the dataset.As a second step in our study, we wanted to assess the possibility of identifying a correlation between the structural similarities of CAIs and their selectivity for a specific hCA isoform over others in an automated way, allowing the development of a protocol exclusively based on ligands’ structures, without taking into account any kind of information concerning the structure of the target proteins. Due to the recently emerged interest in hCA IX as a new potential target for the treatment of hypoxic tumors, we decided to use hCA IX as a test target for this analysis, while considering the ubiquitously expressed hCA II as the corresponding off-target over which the selectivity of compounds would have been desirable. Therefore, we selected the 1756 ligands showing Supplementary Figure S1).As shown in on, ligands selective for the off-target hCA II (SI < 0.2) were defined as Loff, while non-selective ligands (0.2 < SI < 5) were defined as Lns. Subsequently, a selectivity score (Sscore) ranging from +100 to −100 was calculated for each cluster based on the distribution of Lon, Loff, and Lns compounds therein included . An Sscore value of +100 would be obtained for a cluster populated only by Lon ligands, while clusters including only Loff ligands would show an Sscore value of −100. Clusters containing Lon and Loff ligands in similar number and/or including a high number of Lns compounds would show Sscore values close to zero. This analysis highlighted a correlation between the selectivity profile of hCA ligands and their structural features, suggesting the possibility to identify common structural motifs in hCA ligands that could be associated with their selectivity for a desired hCA isoform, thus providing useful guidelines for the design of selective CAIs. For this purpose, we clustered the dataset ligands using orange-canvas software, which performs various methods of hierarchical clustering based on distance metrics. In this case, we performed a single-linkage clustering of the dataset ligands based on their structural similarity described as Tanimoto distances. This analysis was performed considering the Ti values calculated using both FP2 and MACCS fingerprints; for this reason, we generated all possible clusters of ligands with high reciprocal Ti values according to both fingerprint types . The generated clusters were then analyzed based on the selectivity of the ligands included therein. Initially, the compounds were divided into three different classes based on their SI: ligands selective for the on-target hCA IX (SI > 5) were defined as Lscore. Notably, in both cases a considerable number of selective clusters, counting up to 41 compounds and predominantly including either Lon or Loff ligands, were generated. By visually inspecting the compounds populating these clusters it was possible to identify structural elements associated with hCA IX selectivity. score = +100) of hCA IX ligands identified using MACCS fingerprints. All 41 ligands included in this cluster share a sulfamate moiety as ZBG and present a ureidic fragment connected to it through a phenyl ring. All these compounds are endowed with high inhibitory activity against hCA IX, with Ki values ranging from 5 to 100 nM, and an 8 to 80-fold selectivity for hCA IX over hCA II. Compound 1 . Therefore, it appears that this particular structural motif would be able to confer the desired selectivity profile to new potential CAIs designed based on the same scaffold. Our selectivity profile analysis was also suitable for identifying structural motifs shared by promiscuous ligands, which should be thus avoided in the design of selective CAIs and taken into account only for the design of ligands targeting multiple hCA isoforms (in this case hCA IX and II). A remarkable example of a completely non-selective cluster (Sscore = 0) is represented by a small set of ligands with a well-defined structure, bearing a phenylsulfonamide head and an N-tert-butoxycarbonyl tail connected by an alkoxy linker. The compounds belonging to this cluster show double-digit nanomolar Ki values for both hCA IX and II and completely lack of any selectivity. For instance, compound 2 , thus completely populated by Lon ligands (selective for hCA XII over hCA II). Interestingly, the sulfamate inhibitors included in the above mentioned cluster of hCA IX selective compounds .As a further evaluation of our selectivity profiling platform, we repeated the whole analysis considering hCA XII as the on-target and maintaining hCA II as the off-target. For this purpose, we selected a dataset of 1388 ligands showing ompounds  were alsKi values for both hCA II, IX and XII, and repeated the whole analysis. In this case, only MACCS fingerprints were used, since they demonstrated to perform better than FP2 for clustering purposes in the previous analyses, generating clusters of ligands with a more defined central scaffold shared by all compounds. Moreover, for each generated cluster of compounds, we separately calculated two different Sscore values relative to hCA II/IX and hCA II/XII selectivity. The cluster evaluation was then performed based on the analysis of the two Sscore values. The most interesting clusters were visually inspected in order to identify common structural motifs already highlighted in the previous analysis. As expected, the sulfamate ligands represented by compound 1  of +200. Moreover, the analysis identified another small cluster of compounds with high double Sscore, endowed with remarkable activity and selectivity for both hCA IX and XII; these compounds are characterized by the presence of a trifluoroacetyl group and a Y-shaped structure . Conversely, the cluster of ligands represented by compound 2 (score = 0); therefore, these molecules were confirmed to be promiscuous CAIs. The results of these double selectivity analysis demonstrated the reliability of our protocol in identifying ligands with selectivity for multiple hCA isoforms at the same time.Based on these results, we thought about the possibility of applying the same procedure to identify ligands selective for more than one hCA isoform at the same time. For this reason, we selected the 1322 compounds showing mpound 1  were cortructure , as obsempound 2 , was fouKi values for the different hCA isoforms, as previously employed, which should be selected from the File Reader node. Subsequently, it is enough to set the two String Input nodes, specifying the on-target and off-target that should be considered for the selectivity profile analysis, and to execute the workflow. All operations required for the analysis, including fingerprint generation, hierarchical clustering and calculation of Sscore for the obtained clusters, are sequentially performed through multiple KNIME nodes, grouped into a central meta node, as soon as the workflow is started . The results of the analysis can be checked through two different output tables that are automatically produced at the end of all calculations: a Microsoft Excel spreadsheet and a MarvinView table that allows to visualize the results as in Instant JChem software, both including 2D structures, identification codes and bioactivity information for the ligands belonging to clusters with Sscore >70, thus selective for the on-target.With the aim of automatizing our protocol for selectivity profile analyses and make it accessible even to the non-expert user, we developed a KNIME Analytic Platform workflow combining all the different steps included in the protocol. The developed workflow, shown in http://www.mmvsl.it.The selectivity for a desired hCA isoform is a pivotal feature that must be taken into account when developing novel hCA ligands with the aim of obtaining valuable therapeutic tools for the treatment of a desired pathology, with low probability of showing adverse effects due to residual activity against off-target CAs. In the present study, we developed an efficient protocol that allows to analyze the selectivity profile of the different CAIs reported in literature. By using in-house python scripts, we were able to obtain a comprehensive dataset of CAIs including ligand structures and bioactivity data for the different hCA isoforms, which were retrieved from the publicly accessible PubChem database. The dataset was then used to develop and validate a chemoinformatic platform for selectivity profile analyses exclusively based on the comparison of ligand fingerprints and bioactivity data, thus requiring neither information about the structure of the target proteins nor about the binding mode of the analyzed ligands. Our platform allows an ultrafast ligand-based analysis that highlights the presence of structure-selectivity relationships, i.e. the identification of common structural motifs shared by ligands endowed with a similar selectivity profile. In the context of the case studies herein reported, the platform was able to identify specific clusters of ligands with high activity and selectivity for either hCA IX or hCA XII over hCA II, as well as compounds with double selectivity for hCA IX and XII over hCA II, thus providing useful guidelines for the design of selective inhibitors of the tumor-related hCA isoforms. At the same time, the platform highlighted the presence of completely non-selective clusters of compounds with similar activities against the three hCA isoforms, thus also demonstrating its efficacy in identifying useful structural motifs for the potential design of pan-selective compounds. The developed platform was implemented as a KNIME workflow, thus allowing an ultrafast selectivity profile analysis that can be performed in just few clicks and be even accessible to non-expert users. The platform is applicable for selectivity profile analyses focused on all types of ligands and all kinds of protein targets for which a considerable number of bioactive compounds is available in PubChem database. The platform will be made available to all users as a toolbox on our research group website: Click here for additional data file.


The plasticity of the individual epigenetic landscape that goes to countless rearrangements throughout life is closely the reflection of environmental factors such as chemical exposure, socio-economic status and nutrient intakes both early and late in life. The Mini Nutritional Assessment (MNA) is a well-validated tool for assessing malnutrition in old people. It includes 6 (MNA-SF) or 18 (MNA-LF) self-reported questions derived from general, anthropometric, dietary, and self- assessment. We evaluated the association between the nutritional status, as measured by MNA, and methylation biomarkers we previously demonstrated to be associated with chronological and biological age in human. We found that malnutrition is positively correlated with DNA methylation status at the global level, in line with our previous reports. On the contrary, most of the sites located within specific genes, which were previously reported to be correlated with chronological and biological aging, showed to be not affected by malnutrition, or even to have correlations with malnutrition opposite to those previously reported with frailty. These results may suggest that malnutrition is among the first effects of disability and other age- related problems and a generalized non-specific epigenetic remodeling may be the initial response of the organism. By contrast, the fine remodeling of specific genomic sites is scarcely affected by malnutrition and may respond to a more complex interaction of different factors. Therefore, although malnutrition in the elderly is certainly a risk factor for survival, this is partially independent of the aging process of the organism which leads to the methylation remodeling previously described to measure chronological and biological aging. The multi-causal phenotypic variability among elderly individuals has led researchers to evaluate changes in a wide variety of biological parameters in order to have a more comprehensive insight of health at old age and, possibly, predict both the quality of aging and the onset of age-related diseases , 2. In tin utero, energy-rich, protein-deficient, micronutrient-deficient, and/or methyl donor-rich diets induce multi-tissue perturbations of methylation profile in mothers , a validated assessment tool to measure nutrition status in elderly people, and previously identified age-related methylation biomarkers at both nuclear and mitochondrial level.Socio-demographic characteristics of the population sample analyzed in this study were previously reported . BrieflyYounger subjects were contacted through general physicians. Finally, each subject was recruited after a complete multidimensional geriatric assessment with detailed clinical history, including anthropometric measures and a set of the most common tests to assess cognitive functioning, functional activity, physical performance, and depression. In addition, common clinical hematological tests were performed. White blood cells (WBC) from blood buffy coats were used as source of DNA.Vital status at near 9 years from the baseline visit was traced for 189 subjects (98.95%) in S1 and for all the 111 subjects (100%) in S2 through the population registers of the municipalities where the respondents lived.The Mini Nutritional Assessment is a well-validated tool for assessing malnutrition in old people . It inclMT-RNR1) gene, 4 CpG units  falling into the promoter of the ribosomal RNA genes (rDNA), and 12 CpG sites  located within promoter regions of genes involved in mitochondrial quality control  .t-test for continuous variables and chi-square for categorical ones.We compared S1 and S2 groups with regard to study variables and covariates. We used the unpaired For each group, Kaplan-Meier survival curves were estimated for the MNA risk categories (MNA-SF < 12 and MNA-LF < 17 or MNA-LF < 23.5). In order to evaluate their predictive values with respect to mortality risk, the obtained survival curves were then compared by log-rank test. Subjects alive after the follow-up time were considered as censored, and this time was used as the censoring date in the survival analyses. In addition, Hazard ratios (HR) and 95% Confidence Intervals (95% CI) were estimated by using Cox proportional hazard models taking also into account possible confounder variables (age and gender).A linear regression model was used to assess the association between the variability of epigenetic markers and MNA scores. Analyses were adjusted for age at the recruitment and gender.All statistical analyses were performed using IBM SPSS statistics for Windows, V.25 (IBM Corp).Subjects younger and older 85 years of age were included in the study. P = 0.001). Among the subjects who obtained a screening score lower than 12 in S1 group, we found that after the follow-up period, all subjects with severe malnutrition (MNA-LF < 17) died (panel C), compared to 56.0% of those who were at risk for malnutrition (MNA-LF > 17 and MNA-LF < 23.5) and 10.0% of those who were not at risk (MNA-LF>23.5) (P = 0.002).P = 0.177). As it regards MNA-LF, we found that malnourished subjects (MNA-LF < 17) had a significantly increased risk of mortality with respect to subjects with a normal nutritional status  as well as those with a nutritional status at risk , also after adjusting for age and gender.To assess the independent predictive validity of malnutrition in terms of survival in S1 group, we evaluated its association with prospective mortality risk by Cox proportional hazard models. After adjusting for age at recruitment and gender the association between MNA-SF and mortality risk did not hold statistical significance . No significant correlations between MNA-SF and epigenetics biomarkers were detected in S2 group . No significant correlations between MNA-SF and epigenetic biomarkers were detected in S2 group .Among the subjects who obtained a screening score lower than 12 in S1 group, we found that after adjusting for age at recruitment and gender, RAB32_Amplicon1_CpG_24 methylation levels were significantly correlated with the MNA-LF scores . However, this correction seems more suitable when searching for associations without a priori hypotheses but too conservative when assessing a specific research question, as in our case, where a number of non-independent tests  were also performed  are associated with malnutrition. This adds new data in the already well-demonstrated relationship between DNA methylation and frailty . In factOn the contrary, most of the sites located within specific genes, which were previously reported to be correlated with chronological and biological aging, showed to be not affected by malnutrition. Moreover, the few methylated sites associated with MNA scores showed correlations not immediately in line with previous results. Indeed, we found a direct correlation between a good nutritional status and the methylation levels of rDNA_CpG_5 and RAB32_Amplicon1_CpG_24, while we have previously reported that high proportion of the methylation of these sites is correlated with impaired cognitive performance, decreased survival chance and disability , 58. TheThe above associations are restricted to the subjects of S1 group (<85), possibly because malnutrition may need some time to act on methylation and the higher mortality of the subjects older than 85 years (S2 group) may lead to death very soon after the start of malnutrition without the time to establish a methylation remodeling of these sites.Finally, it is likely that genetic variants impairing nutrient intake and/or metabolism which could have a role in the interplay we observed and create an additional layer over epigenetics changes.We are aware that our study has some weaknesses that should be addressed. A first limitation of the study is the reduced sample size. Its cross-sectional design does not allow us to assess the cause-effect relationship between the variability of epigenetic markers and the nutritional status of subjects under study. Another important limitation is the lack of proper correction for multiple testing. However, since this study was exploratory and hypothesis-driven, a Bonferroni correction would have eliminated potentially important findings if applied , 60. ForThe finding of the influence of nutrition on frailty through epigenetic modifications appears particularly relevant because of a balanced nutritional intervention could be an easy to use clinical approach for population-based strategies in aging to provide favorable functional and mortality outcomes and, at the same time, minimize the hospital assistance and long-term care, thus reducing the health care costs.The datasets generated for this study are available on request to the corresponding author.PD'A, AM, GP, and DB designed the study. AM and VR performed the statistical analysis. DB and GP wrote the initial draft. AM, PD'A, VR, GP, and DB participated in critical revision and approved the final manuscript before submission.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.


Caenorhabditis elegans. Mutants of the CRL4 complex  show defects in SC assembly manifested in the formation of polycomplexes (PCs), impaired progression of meiotic recombination, and reduction in crossover numbers. PCs that are formed in cul-4 mutants lack the mobile properties of wild type SC, but are likely not a direct target of ubiquitination. In C. elegans, SC assembly does not require recombination and there is no evidence that PC formation is regulated by recombination as well. However, in one cul-4 mutant PC formation is dependent upon early meiotic recombination, indicating that proper assembly of the SC can be diminished by recombination in some scenarios. Lastly, our studies suggest that CUL-4 deregulation leads to transposition of the Tc3 transposable element, and defects in formation of SPO-11-mediated DSBs. Our studies highlight previously unknown functions of CRL4 in C. elegans meiosis and show that CUL-4 likely plays multiple roles in meiosis that are essential for maintaining genome integrity.To maintain the integrity of the genome, meiotic DNA double strand breaks (DSBs) need to form by the meiosis-specific nuclease Spo11 and be repaired by homologous recombination. One class of products formed by recombination are crossovers, which are required for proper chromosome segregation in the first meiotic division. The synaptonemal complex (SC) is a protein structure that connects homologous chromosomes during meiotic prophase I. The proper assembly of the SC is important for recombination, crossover formation, and the subsequent chromosome segregation. Here we identify the components of Cullin RING E3 ubiquitin ligase 4 (CRL4) that play a role in SC assembly in  Caenorhabditis elegans) that lack protein components of the Cullin 4 E3 Ubiquitin ligase complex (CRL4), have defects in the formation of the SC that can be due to misfolding of SC proteins and their aggregation. CRL4 appears to be involved in other germline functions that directly affect chromosome stability (DNA damage repair and transposition), indicating that CRL4 has a central function in the formation of functional sperm and egg cells.Defects in the formation of the structure named the synaptonemal complex (SC) lead to the missegregation of chromosomes in the divisions that generate sperm and egg cells. In humans, this chromosome missegregation is associated with infertility and developmental disabilities of the surviving progeny. Abnormal SC structures composed of misfolded and aggregated SC proteins are associated with an inability to properly repair DNA damage and accurately segregate meiotic chromosomes. How SC proteins assemble such that they do not form misfolded protein aggregates is poorly understood. The germlines of nematodes ( Caenorhabditis elegans, homologous chromosomes pair, synapse, and proceed through meiotic recombination . Prioewed in: ). Synapsewed in: ). SC asssophila;  and C. eelegans; ). In oth. mouse;  and S. cevisiae; ). Negatie.g., . These PCs may be due to SC assembly defects suggesting a role for CRL4 in SC assembly, consistent with  show very limited PC formation that is only significant at 25°C (cul-4(ok1891)/+ likely produce less CUL-4 protein product than +/+ (wild type), the appearance of PCs at 25°C can be attributed to the reduction in protein levels. These data indicate that PCs can be formed due to a reduction in CUL-4 protein levels, and not only through perturbations of CUL-4 protein function such as the gain of function activity of the cul-4(ok1891) allele.Based on fluorescence analysis, the level of CUL-4 protein in the  to 20°C . If cul- at 25°C . Since cddb-1(tm1769) and gad-1(ok573) mutants at 15°C. These two mutants were completely penetrant at 20°C, but had decreased penetrance of PC formation when grown at 15°C from L4 to the adult developmental stage. These data altogether indicate that perturbation of CRL4 activity correlates with defects in PC formation in a temperature-dependent manner.The correlation between temperature and PC formation may be specific to the perturbation of CUL-4 activity, and not the CRL4 complex. To examine if other CRL4 mutants exhibit temperature-dependent PC formation as well, we have grown et al, 2017). The fact that the penetrance of PC formation phenotype is enhanced by increased temperature suggests that the PCs found in CRL4 mutants may be due to SYP protein misfolding, which would lead to loss of the liquid-like properties of the PCs in these mutants. There is evidence that misfolded PCs cannot be disassembled with 1,6-Hexanediol, while properly assembled SC can  do not trigger larval arrest, indicates that this malformed CRL4 complex maintains partial activity circumventing the essential function of CUL-4 during larval development. It is possible that in the absence of the lysine modified by neddylation in wild type CUL-4, another lysine serves as an alternative site for modification in cul-4(ok1891) mutants, rendering it partially active. It is perplexing how CUL-4 protein that lacks the C’ domain (RBX/ROC-E2 binding) was able to bypass the need for a functional CRL4 in some worms. It is possible that the E2 enzyme can modify some CRL4 substrates without directly binding to CUL-4; the C’ truncated form of CUL-4 that is still able to bind the substrate may provide some assistance to the E2 creating a partial loss of function phenotype.The cul-4::pole-1 3’UTR have similar increased levels of RAD-51 as to these observed in the csn-5, ddb-1, and gad-1 mutants. However, in cul-4(ok1891) mutants, meiotic prophase I nuclei have SPO-11 independent DSBs as well as nuclei that lack SPO-11 DSBs. Moreover, in cul-4::pole-1 3’UTR mutants, PC formation is independent of SPO-11, whereas in cul-4(ok1891) mutants PC formation is dependent on SPO-11. These findings indicate that germline nuclei of these two different cul-4 mutants did not produce the same defects, aside from PC formation, and this can be explained by a gain-of-function in the cul-4(ok1891) allele. The gain-of-function phenotypes in cul-4(ok1891) mutants may be due to the formation of an aberrant CRL4 complex  that interrupts SPO-11 induced DSB formation. Alternatively, the unique phenotypes for cul-4(ok1891) mutants compared to cul-4::pole-1 3’UTR mutants may be due to a manifestation of a more severe reduction in CUL-4 levels. In this model, CUL-4 forms a complex with other CRL4 complex proteins, beside GAD-1/DDB-1, and performs germline functions outside SC assembly. In this case, CUL-4 activity in SC assembly (GAD-1/DDB-1 dependent) is perturbed due to the reduction of CUL-4 availability, however other CUL-4 activities such as DSB -dependent PC formation and transposition, are only abrogated when CUL-4 levels drop even further. This may explain why cul-4::pole-1 3’UTR mutants (~70% of wild type protein levels) are similar to ddb-1 and gad-1 mutants, while cul-4(ok1891) mutants (~15% of wild type protein levels) exhibit distinct phenotypes  mutants particularly in comparison to csn-5, ddb-1, and gad-1 mutants is the incomplete penetrance of the PC formation phenotype. PCs were present in 2 out of 3 germlines analyzed and two germlines of the same worm may exhibit different phenotypes. The high variation in levels of nuclear localization of CUL-4 in cul-4(ok1891) mutants may explain the incomplete penetrance observed at 20°C. Similarly, cul-4::pole-1 3’UTR mutants also shows partial penetrance, but is observed at higher temperatures (22–24°C). In cul-4::pole-1 3’UTR mutants, CUL-4 levels are reduced but not to the same extent as cul-4(ok1891) mutants. This may explain why PC formation only appears at higher temperatures in cul-4::pole-1 3’UTR mutants.Another unique feature of C. elegans, the SC assembles along homologous chromosomes similar to wild type  ; LGII: cul-4(ok1891), mIn1 [mIs14 dpy-10(e128)] II; LGIV: csn-5(ok1064), ddb-1(tm1769), msh-5(me23), rad-51(ok2218), spo-11(ok79), spo-11(iow110), nT1[qIs51] ; LGV: cand-1(tm1683), gad-1(ok573), rbx-1(ok782), nT1[qIs51] .The following transgenic lines were used: syp-4(iow28[V5::syp-4]) I, cul-4(iow64[cul-4::pole-1-3’UTR]) II, meIs8[pie-1p::GFP:: cosa-1+unc-119(+)] II, meIs9[unc-119(+) pie-1promoter::gfp::SYP-3];unc-119(ed3) III, syp-3(iow69[3xFLAG::syp-3]) III, ddb-1(iow60[OLLAS::ddb-1]) IV, gad-1(iow61[3xFLAG::gad-1]) V, syp-1(iow68[syp-1::3xFLAG]) V, syp-2(iow27[syp-2::V5]) V.dpy-10 co-injection marker was used as a measure of successful Cas9 excision. CRISPR/Cas9 was performed as described in .All CRISPR/Cas9 reactions used ssODNs and crRNAs generated by Integrated DNA Technologies (IDT), materials used are listed below in the table. Recombinant Cas9 protein was isolated by QB3 MacroLab . A ribed in (with sliribed in . spo-11 ed as in  but by iakir-1(gk528);rrf-1(pk1417) double mutant background. Previous work in our lab has shown that akir-1(gk528) mutants can be used as a sensitized background to find genes that are involved in SC assembly [rrf-1 is a gene encoding an RNA dependent RNA polymerase expressed in somatic tissue; rrf-1 mutants were used to direct RNAi knockdown specifically to the germline, as these RNAi targets likely have a function in somatic tissue. A gfp::syp-3 transgenic line was crossed into these double mutants to quantitatively determine if each knockdown condition influenced SC assembly. Germlines were examined for PC formation in pachytene; PCs were defined as SC structures that are at least two times the width of linearized SC. In this screen, we exposed our double mutants with the gfp::syp-3 transgenic line to RNAi by feeding and performed whole worm ethanol fixation for analysis. RNAi clones were inoculated overnight in Lauria broth and ampicillin (50 ng/μl). Bacterial cultures were seeded on plates containing IPTG then allowed to grow for a minimum of 12 hrs. at 37°C. RNAi clones from the Ahringer C. elegans RNAi library were used [pL4440 empty vector was used as a control. Once the next generation reached L4 stage, they were moved to another set of RNAi seeded plates. The next generation at L4 stage were further moved to another set of plates and as day 1 adults were ethanol fixed and examined for PC formation.To identify possible adapter proteins and E2 conjugating enzyme(s) involved in SC assembly, we performed RNAi knockdown of 41 different possible adapter proteins, containing the DDB1 binding WD40 domain (DWD), and 22 predicted E2 enzymes. The screen was performed in an assembly ,50. rrf-ere used . L1 synccsn-5(ok1064), cand-1(tm1683), rbx-2(ok1617), spo-11(ok79), rad-54(ok615), msh-5(me23), and rad-51(ok2218) strains were dissected 20–24 hrs. post-L4. ddb-1(tm1769), gad-1(ok573), and rbx-1(ok782) strains were dissected 30–35 hrs. post-L4. The cul-4(ok1891) strain was dissected 48–52 hrs. post-L4. Immunofluorescent staining antibodies used are as follows: α-SYP-1 , α-RAD-51 , α-HTP-3 , α-HIM-8 , α-FLAG , and α-OLLAS . Secondary antibodies used were α-goat Alexa Fluor 549, and α-rabbit Alexa Fluor 488. All gonads were stained with DAPI  for 10 min. Immunostaining was performed as in [Adult hermaphrodites were dissected, genotypes were dissected at different times, based on growth rate, after L4 stage. Wild type,  rad-51ok18 strainn reflects the number of either germlines or nuclei analyzed.  C- 10 germlines for all genotypes analyzed; D- 1,712 nuclei; E- 695 nuclei; F- 755 nuclei; G- 657 nuclei; H- 619 nuclei; I- 711 nuclei; J- 1,194 nuclei; K- 1,613; from 3 independent germlines for each genotype. K- wild type 28, csn-5(ok1064) 39, cul-4(ok1891) 39, ddb-1(tm1769) 29, gad-1(ok573) 23, rbx-1(ok782) 27, rbx-2(ok1617) 35, rbx-1(ok782);rbx-2(ok1617) 25, cand-1(tm1683) 21 germlines analyzed. L wild type 189 cul-4(ok1891) 162 SC and 47 PC nuclei,  A- wild type 50, flag::cul-4 70, flag::cul-4(ok1891) 90, and flag::cul-4::pole-1 3’UTR 70, B- From left to right: wild type 24, 38, 12, 16,13 and 8 gonads, flag::cul-4::pole-1 3’UTR 27, 14, 12, 8, 28 and 14 gonads. C- wild type 711 nuclei, cul-4(ok1891) 300 nuclei, D- wild type 15, 24 and 9 germlines, cul-4(ok1891) 15, 39 and 20 germlines, cul-4(ok1891)/+ 21 and 23 germlines, ddb-1(tm1769) 34 and 29 germlines, gad-1(ok573) 5 and 23 germlines,  B- wild type 866 nuclei, csn-5(ok1064) 592 nuclei, cul-4(ok1891) 436 nuclei, ddb-1(tm1769) 502 nuclei; D- wild type 907 nuclei, csn-5(ok1064) 625 nuclei, cul-4(ok1891) 532 nuclei, ddb-1(tm1769) 346 nuclei from 3 independent germlines for each genotype.  A- 1,124 nuclei; B- 552 nuclei; C- 665 nuclei; D- 627 nuclei; E- 1,396 nuclei; F- 755 nuclei; G- wild type 1,124 nuclei, csn-5(ok1064) 552 nuclei, cul-4(ok1891) 665 nuclei, ddb-1(tm1769) 515 nuclei, and rad-54(ok615) 812 nuclei from 3 independent germlines G 100 nuclei, H- 100 nuclei, I- 10 nuclei per genotype.  A- 1,124 nuclei; B- 665 nuclei; C- 1,133 nuclei; D- 422 nuclei; E- 1,444 nuclei; F- 560 nuclei; G- 1,701 nuclei; H- 431 nuclei from 3 independent germlines; I- Between 30 and 70 worms were used for each sample, 4 biological replicates with 3 technical replicates were analyzed for each genotype.  B- wild type 39, csn-5(ok1064) 60, cul-4(ok1891) 83, ddb-1(tm1769) 43, gad-1(ok573) 55, cand-1(tm1683) 89, rbx-1(ok782) 79, rbx-2(ok1617) 79 nuclei were analyzed from 3 independent germlines; D- wild type 22, rbx-1(ok782) 20, rbx-2(ok1617) 21, cand-1(tm1683) 20 diakinesis-1 nuclei were analyzed.  A- 1,712 nuclei; B- 657 nuclei; C- 1,694 nuclei; D- 439 nuclei; E- 1,629 nuclei; F- 501 nuclei; G- 1,646 nuclei; H- 701 nuclei; I- 1,399 nuclei; J- 610 nuclei; K- wild type 28, cul-4(ok1891) 39, spo-11(ok79) 22, cul-4(ok1891);spo-11(ok79) 23, rad-51(ok2218) 23, cul-4(ok1891);rad-51(ok2218) 28, rad-54(ok615) 20, cul-4(ok1891);rad-54(ok615) 33; msh-5(me23) 27; cul-4(ok1891);msh-5(me23) 30, csn-5(ok1064) 39, csn-5(ok1064); spo-11(ok79) 31, ddb-1(tm1769) 29, ddb-1(tm1769); spo-11(ok79) 19, cul-4::pole-1-3'UTR; spo-11(ok79)/nT1 18 23°C, cul-4::pole-1-3'UTR; spo-11(ok79) 28 23°C, cul-4::pole-1-3'UTR; spo-11(ok79)/nT 11 25°C, cul-4::pole-1-3'UTR; spo-11(ok79) 10 25°C, germlines analyzed.  B through E- at least 3 replicates of each western was performed with whole worm lysate from 30–50 worms per genotype,  wild type 12, cul-4(ok1891) 12 DHC-1 patches; from at least 3 independent germlines per genotype,  A- 627 nuclei; B- 1,328 nuclei; C- 1.592 nuclei; D- 1.570 nuclei; E- 1,493 nuclei; F- 507 nuclei; from 3 independent germlines per genotype.GraphPad Prism was used for generating of all graphs and statistical analyses. Adult hermaphrodites were fixed with ethanol and sealed with DAPI and Vectashield upon reaching day one adult stage. Full worm images were taken with a Leica DM RXA compound light microscope with QI Click Camera 20X lens. Images were analyzed using softWoRx software by calculating the distance between the distal tip cell to the end of pachytene. Ten worms were analyzed per genotype. Statistical comparison: two-tailed Mann-Whitney.cul-4(ok1891) mutants having six zones. Each zone represented a time point in meiotic prophase I progression . Zones were created by imaging from the distal tip cell using a 512x512 pixel dimension frame of view. Images were taken until the end of pachytene.Germlines were divided into zones based on length: wild type length having seven zones and RNAi Screen for PC definition).Adult hermaphrodites were fixed with ethanol and sealed with DAPI and Vectashield upon reaching day one adult stage. Whole worms were observed under the Deltavision System described earlier. Germlines were examined for GFP::SYP-3 PC formation  strains. Superscript III OneStep RT-PCR kit  and primers 5’-ttagctgctttcgagccttc-3’ and 5’-gcttcaaatccgcaaatgat-3’. The obtained RT-PCR fragment was sequenced to reveal a deletion of part of exon 10 and most of exon 11 which created an out of frame deletion (junction sequence: TCTCGATCAAATGGTA/AGAAGGAAGGTACTGTG).RT-PCR was performed in Tc1 and Tc3, TC1/Mariner class DNA transposable elements, in wild type and cul-4(ok1891) strains. Superscript III OneStep RT-PCR kit  and primers designed as in [Tc1: 5’-CTT GAA GCG CTT CTT GTC ACGC-3’ and 5’-CCA ACC ACT GGA ACG ACC GTG-3’; Tc3: 5’-GAG CGT TCA CGG AGA AGA AG-3’ and 5’-AAT AGT CGC GGG TTG AGT TG-3’. The Roche LightCycler 480 Real-Time PCR System was used for qRT-PCR and analysis of Tc1 and Tc3 transcript abundance. Actin RT-PCR was used for normalization: 5’-ATC ACC GCT CTT GCC CCA TC-3’ and 5’-GGC CGG ACT CGT CGT ATT CTT-3’. 30 worms with equal numbers of wild type and cul-4(ok1891) mutants used in each qRT-PCR prep. Each qRT-PCR prep was split into 3 technical controls. For each genotype, 3–4 biological controls were used. Samples were excluded if Cts > 29 for actin or if Ct = 40 for Tc3 in any of the replicas. Each Tc3 sample was normalized to actin and two paired samples were subtracted to get ΔΔCT. Relative expression was obtained from ΔΔCT.Quantitative RT-PCR was performed on ed as in  were usecsn-5(ok1064) mutants this represented micronuclei formation, and in cul-4(ok1891) mutants this represented an EMO-like dispersed DAPI appearance. Statistical comparison: Fisher’s exact test.Qualitative scoring of SYP-1 localization patterning within individual nuclei was determined by placement into one of eight different categories. “No SYP-1” was defined as having no SYP-1 immunofluorescence present along chromosomes (DAPI). “SYP-1 PC” were nuclei with only PC formation present, no elongation of SYP-1 along DAPI. “SYP-1 PC and Some Linear” was the presence of PC(s) in the nucleus but also partial elongation of SYP-1, <50% of DAPI. “SYP-1 PC and Linear” was similar to “SYP-1 PC and Some Linear” with the exception that SYP-1 is elongated along >50% of DAPI. “SYP-1 Partial Linear” nuclei had elongated SYP-1 along up to 50% of DAPI. “SYP-1 Mostly Linear” nuclei had SYP-1 along up to 50% of DAPI but less than 100%. “SYP-1 Linear” nuclei had fully elongated SYP-1 along all DAPI. “Other” was defined as nuclei that had abnormal DAPI appearance, for example in All zones were quantified for number of RAD-51 foci per nucleus as in . Three gRAD-51 Foci Analysis were used. Full nuclear projections were created and analyzed in ImageJ. An ellipse (1 pixel wide) was placed around a targeted nucleus, based on DAPI staining, using the circle tool. Mean intensity of the RAD-51 channel was calculated and divided by the number of RAD-51 foci with that nucleus. These values were then normalized to the mean intensity of the RAD-51 channel in an ellipse placed in the cytoplasm. Statistical comparison: two-tailed Mann-Whitney.16-bit non-deconvolved images acquired with a one second exposure were analyzed by FIJI software. Intensity measurements using anti-FLAG antibody were gathered by drawing a circle around a nucleus at its greatest width from a single Z-stack from PMT or LP. For each genotype and region, 10 nuclei per germline were measured for three or more germlines. Background was recorded at 4 different positions inside the cytoplasmic space and the average of the cytoplasmic intensities was subtracted from each nuclear intensity recording. Mann-Whitney U test (GraphPad Prism 7 software) was used to statistically compare intensities of staining. For RAD-51 intensity analyses, images acquired for RAD-51 Foci Analysis were used. Individual nuclei were analyzed for abnormal RAD-51 appearances. Abnormal RAD-51 were defined as anything that did not form wild type puncta. Examples of this include stretches of RAD-51 or a globular “lobed” appearance larger than wild type puncta. Statistical comparison: Fisher’s exact test.Images acquired for Immunofluorescent Staining and Microscopy after L4 stage selection. Dissected germlines were incubated in 15% 1,6-Hexanediol then fixed in 3.7% PFA. Germlines were then immunofluorescent antibody stained for SYP-1 and HTP-3. Using the DeltaVision system and softWoRx software, images were analyzed for presence of SYP-1 PC formation and linearization along the axial element HTP-3. Statistical comparison: Fisher’s exact test.Adult worms were dissected in Egg buffer as in timeline for different mutant strains described in Localization Analysis, was used to divide germlines and were scored for 5S rDNA FISH signal/HIM-8 foci pairing in each nucleus. Paired foci were determined as being 0.7-μm or less apart [Zonal division, as in SYP-1 ss apart . Statistss apart .GFP::COSA-1 was quantified in the final zone of each germline (either Zone 6 or 7 dependent upon germline length), corresponding to late pachytene . COSA-1 Images were taken of the final diakinesis nucleus (diakinesis -1) prior to spermathecal entrance. Number of DAPI bodies were totaled following counting in 3D. Statistical comparison: two-tailed Mann-Whitney.cul-4(ok1891) mutant background. Images of single plane at TZ (based on distance from tip and presence of DHC-1::GFP foci) were taken for 60 s. Analysis was performed in ImageJ using the plugin Manual Tracking for tracking of DHC-1::GFP foci. Data points were taken at the onset and conclusion of each directional movement, obtaining the calculation of total distance traveled, mean velocity, number of times foci changed direction, and time between movements of foci. All DHC-1::GFP foci visualized during the whole duration of the movie (60s) were analyzed. Statistical comparison: two-tailed Mann-Whitney.Analysis of movement was performed in DHC-1::GFP transgenic strains in the wild type (N2) and Strains are available upon request.S1 Figcul-4(ok1891) mutant background. Results are grouped into three categories: whole worms with no PC formation in either germline, worms with PC formation in one germline, or worms with PC formation in both germlines. The number of germlines with PCs is not significantly different from observations in SYP-1 immunofluorescent analyses. Scale bars are 2μm. K-N) Immunostaining using antibodies against HTP-3 (green) and SYP-1 (red) in CRL mutants shows that HTP-3 localized to chromosomes in linear fashion about the ame time SYP-1 appears, in all genotypes analyzed. Scale bars are 5μm.A, B, & D-J) Representative images of SYP-1 immunofluorescent staining in CRL4 mutants throughout progression of the germline. Blue (DAPI) and red (SYP-1). Scale bars are 2μm. C) Table representing analysis of GFP::SYP-3 in the (TIF)Click here for additional data file.S2 Figddb-1::OLLAS transgenic line. B) Representative images of immunofluorescent staining against FLAG is presented as progression through meiotic prophase I of the e gad-1::FLAG transgenic line. C) Representative images of immunofluorescent staining against FLAG in late pachytene of the FLAG::cul-4 transgenic line. Circles indicate nuclei, as an example of the quantification in A) Representative images of immunofluorescent staining against OLLAS is presented as progression through meiotic prophase I of the (TIF)Click here for additional data file.S3 Figsyp-1::FLAG; syp-2::V5; FLAG::syp-3; V5::syp-4) was performed with whole worm lysates. Expected sizes for SYPs are: SYP-1 56.6 kDa, SYP-2 23.7 kDa, SYP-3 25.8 kDa and SYP-4 67.3 kDa. See cul-4(ok1891) mutant background but this was not repeated in 2 other blots. For G and I the same wild type control is used. K is quantification of standard western blots, while L is quantification with proteasome inhibition. For K and L all replications were included (n of at least 3 western blots).A-F) Western blot analysis of SYP proteins ((TIF)Click here for additional data file.S4 Figcul-4(ok1891) mutants in TZ nuclei shows no requirement for CUL-4 in chromosome movement.A-I) Representative images of SUN-1 immunofluorescent staining in CRL4 mutants in TZ and LP, genotypes indicated on the side. Blue (DAPI) and green (SUN-1). SUN-1 patches are present in all genotypes at TZ, but some LP nuclei contain patches as well in CRL4 mutants that form PCs or show accumulation of recombination intermediates . Scale bars are 2μm. J-M) analysis of movement of DHC-1::GFP foci in wild type and (TIF)Click here for additional data file.S5 Figcul-4(ok1891) and ddb-1(tm1769)) mutants  average +/- SEM.A-D) Left: representative images of RAD-51 immunofluorescent staining in CRL4 E3 ligase mutants. Blue (DAPI) and green (RAD-51). Right: graphical analyses of RAD-51 foci appearance throughout the germline. Statistical comparisons were compared to wild type worms . E & F) Left: representative images of RAD-51 immunofluorescent staining. Right: analyses of number of RAD-51 foci per nucleus throughout meiotic prophase I. Blue (DAPI) and green (RAD-51). Statistical comparisons of double mutants were made against single mutants . Scale bars are 2μm. G) Fold change values in (TIF)Click here for additional data file.S6 FigC. elegans by others [A) SC assembly in the genotype tested, B) the connection between recombination and PC formation in CRL mutants. The structure of the CUL4 complex is based on work in other organisms. Physical interaction between DDB-1 and CUL-4 was shown in y others .(TIF)Click here for additional data file.S1 FileThis file contains the underlying numerical data for all the figures. Each tab corresponds to a figure panel with numerical data.(XLSX)Click here for additional data file.


The impact of integrating video into health education delivery has been extensively investigated; however, the effect of integrating video on a learner’s subsequent performance in an online educational setting is rarely reported. Results of the relationship between the learner’s online video viewing and subsequent progression toward health behavior change in a self-directed online educational session are lacking.This study aimed to determine the relationship between viewing a Health eKitchen online video and key engagement performance indicators associated with online nutrition education for women, infants, and children (WIC).t tests were performed to test the difference in average link depth and duration of use between groups overall and stratified by lesson type. Logistic regression was conducted to validate the impact of video viewing prior to lesson completion while controlling for lesson type and factors known to be associated with WIC key performance indicators.This study involved a retrospective cohort of users grouped on the basis of whether Health eKitchen exposure occurred before or after completing a nutrition education lesson. A two-sample test for equality of proportions was performed to test the difference in the likelihood of progression between the groups overall and when stratified by lesson type, which was defined by whether the lesson focused on food preparation. Welch two-sample 2=12.6, P<.001) and non-food preparation  lessons among early stage users who had viewed a Health eKitchen video before completing a lesson. Time spent viewing educational learning resource links within the lesson was also significantly longer for both food preparation  and non-food preparation  lessons. Logistic regression analysis corroborated these results while controlling for known confounding factors. The odds of user progression were nearly three times greater among those who viewed a Health eKitchen video prior to lesson completion . Type of lesson (food vs non-food preparation) was the strongest predictor of progression odds .A greater stage of change progression was observed for both food preparation (χUser access to a Health eKitchen video prior to completion of an online educational session had a significant impact on achieving lesson goals, regardless of the food preparation focus. This observation suggests the potential benefit of providing an application-oriented video at the onset of online nutrition education lessons. The federally funded Special Supplemental Nutrition Program for Women, Infants, and Children (WIC) provides qualified program participants who are at nutrition risk due to insufficient financial access to nutritious foods to supplement diets, information on healthy eating, and referrals to health care. Many positive health outcomes have been associated with the program, including improved diet, infant feeding practices, and preconceptual nutrition status . WichealThe impact of integrating video into health education delivery has been extensively investigated to determine whether learning objectives can be achieved more effectively and with greater learner satisfaction than interventions that do not use any technology -10. ThisTypical studies concerning the impact of video use in an online learning environment focus on comparing various educational modalities that include video with those that do not or those that have varying levels of interactive video integrated ,16,22. CThe purpose of this study was to determine the relationship between viewing of a Health eKitchen online video and key engagement performance indicators associated with WIC online nutrition education.The sample used for this study was derived from WIC clients across 21 US states, who completed a wichealth.org lesson during a 34-month period in the government’s 2014-2016 fiscal years. Participating subjects chose to complete a wichealth.org lesson as a means of meeting secondary contact requirements associated with the WIC program. Data-collection protocols using wichealth.org were approved for use by the Western Michigan University’s Human Subjects Institutional Review Board. Online informed consent was provided prior to completion of the online survey.Data utilized in this study were collected from 4460 wichealth.org uses from the system database for WIC clients who started an English-based lesson in one of the first three stages of the Transtheoretical Model , completed the lesson, and viewed at least one Health eKitchen video during their session. Only the individuals who began a lesson in the early stages of readiness to change, not those in the active change stages, could have measurable progression at the time of lesson completion.All lessons were completed using the wichealth.org website application. Data consisted of six system-collected measures , four profile items , and time at which the Health eKitchen video was viewed. The staging algorithms used to identify the beginning and ending stages were based on criteria previously used to determine stages of change and progression, which have been described in detail elsewhere ,4.Binary progression—irrespective of whether a subject advanced in stage of change intent—was the dependent variable used. The primary independent variable of interest was subject exposure to a Health eKitchen video prior to completion of a wichealth.org lesson. Over the past decade, program evaluations conducted every 6 months demonstrated the importance of other variables currently collected from users of wichealth.org. These include user demographic characteristics of race, ethnicity, pregnancy status, and age as well as system access characteristics including device type , lesson type (feeding behavior focus vs other), number of internet resource links accessed (depth), and duration of all resource links used. Each of these variables was considered a possible effect modifier or confounder of the relationship of interest. Depth and duration of link use are key performance indicators for wichealth.org previously demonstrated to be strongly associated with users’ stage of change progression . Lesson In order to control for motivation bias potentially confounding the positive relationship between lesson engagement and viewing a Health eKitchen video prior to wichealth.org lesson completion, users who chose to view a Health eKitchen video after completing a wichealth.org lesson were used as the comparison group. Similar to users who viewed a Health eKitchen video prior to their lesson, these individuals were motivated to view a video, but they viewed the video after their lesson. As users access Health eKitchen by their own choice and motivation, it is likely they are motivated to be engaged to a greater extent and subsequently more likely to progress in the stage of readiness change compared to users that opt not to access Health eKitchen at all.t tests were performed to test whether link depth and duration were associated with user progression and each of the covariates. The Welch t test adjusts the number of degrees of freedom when the variances are not equal between groups. A logistic regression was conducted to evaluate the odds of progression for users who viewed a Health eKitchen video prior to subsequent completion of a wichealth.org lesson compared to those who did not. All data were analyzed using the R statistical package [A two-sample test for equality of proportions was performed to test the difference in the likelihood of progression between comparison groups. Likewise, this difference was tested for each of the covariates: Welch two-sample  package .P<.001; Approximately half  of the subjects included in this investigation reported that they were of Latino ethnicity, and 58.5% of subjects belonged to a race other than white. Most  subjects were mothers of the child who was receiving WIC benefits. In addition, 2655 of the subjects (59.53%) completed the lessons using a fixed device. As expected, device type and lesson type were both significantly associated with the outcome of interest or at least one of the wichealth.org key performance indicators P<.001; . Further2=127.2, P<.001; t=8.2, P<.001). In addition, both food preparation  and non-food preparation  lessons demonstrated significantly greater progression among early stage of change subjects who viewed a Health eKitchen video prior to the lesson. Similarly, the average time spent was significantly longer for both food preparation  and non-food preparation  lessons among users who viewed a Health eKitchen video prior to the lesson.The wichealth.org stage of change progression (one or more stages) was significantly associated with whether a user accessed a Health eKitchen video prior to their lesson (χOnly predictors significantly associated with the odds of stage progression were included in the model . AlthougVideo use has been well demonstrated as a valuable and impactful tool in the online educational setting. This study evaluated the effect of video viewing on intent to change parent-child feeding behavior when the video was viewed before beginning the educational lesson. Potential for confounding caused by motivation or volunteer bias associated with learners who chose to view a Health eKitchen video prior to their lesson was minimized, as the comparison group also chose to view Health eKitchen videos but did so after completion of their educational session.The results of this study are important, because even though online nutrition education programs have been found to have an impact on various aspects associated with nutrition behaviors among WIC clients, including belief in ability and intent to positively impact parent-child feeding, knowledge and behaviors surrounding eating breakfast, and reduction in salt intake -5,26,27,Findings from this investigation suggest that early stage wichealth.org users were more successful in progressing in stage of behavior change intent when the session focusing on that behavior was preceded by a Health eKitchen video view as compared to when it was followed by video viewing while controlling for other key predictors of progression. It was expected that if an effect was identified, it would be limited to lessons focusing on meal-preparation behaviors, as Health eKitchen videos would mostly be relevant to such behaviors, rather than lessons focusing on, for example, behaviors such as physical activity or dental hygiene. However, users of non-meal preparation lessons benefited to a similar extent as users of meal preparation lessons when they used Health eKitchen before completing their lesson, suggesting that the content of the video may be less important than the arousal or level of engagement that may be incited in the learner following video viewing. A previous study on the use of video and its ability to enable deep learning that touched on the subject reported that the relevance of the video with a learning objective should be carefully considered and clear to the user . In contSubjects chose whether they wanted to use wichealth.org as the method for completing secondary contact requirements, which limits the ability to generalize the results of this study to the entire WIC population. In addition, Health eKitchen is a separate feature that does not qualify for secondary contact credit. As such, the two groups compared in this study may differ in how they approach educational learning. Nonetheless, the use of video prior to initiating an online learning session appears to be an important strategy to employ in e-learning environment designs in order to maximize user engagement and subsequent achievement of learning objectives.We recommend that a more rigorous study be conducted to confirm our findings and allow generalization of the results to a broad audience of WIC participants. Additional studies could focus on introducing video learning in other online education and behavior change programs used by WIC to determine if our findings are applicable to any online learning approach with WIC clients or specific to the Health eKitchen feature available within the wichealth.org nutrition education platform. The use of video prior to the use of other traditional educational formats available within WIC clinics, such as information malls and one-on-one nutrition counseling, could be explored as a means for potentially impacting client engagement. Many clinics provide kiosks or looping videos in clinic waiting areas where nutrition-related videos could be shown. Although not the same as an online format, the implication that the use of video may, to some degree, influence client engagement warrants exploration of this method of learning.This study is useful to nutritionists and WIC staff for developing or adopting online nutrition education and behavior change interventions, especially for WIC populations. This study showed that viewing a video prior to lesson completion was positively related to subject engagement in the online educational session.


Within qualitative research in-person interviews have the reputation for being the highest standard of interviewer-participant encounter. However, there are other approaches to interviewing such as telephone and e-mail, which may be appropriate for a variety of reasons such as cost, time and privacy. Although there has been much discussion of the relative values of different interview methods, little research has been conducted to assess what differentiates them using quantifiable measures. None of this research has addressed the video call, which is the interview mode most like the in-person interview. This study uses quantifiable measures generated by the interview to explore the relative value of in-person and video call interview modes.Interview data gathered by a qualitative research study exploring the views of people with IBS about hypnotherapy for their condition were used. In-person and video call interviews using the same topic guide were compared on measures of length (time and word count), proportion of time the interviewer was dominant, the number of topics generated (codes) and the number of individual statements on which those topics were based.Both interview methods produced a similar number of words and a similar number of topics (codes) were discussed, however the number of statements upon which the variety of topics was based was notably larger for the in-person interviews.These findings suggest that in in-person study interviews were marginally superior to video calls in that interviewees said more, although this was on a similar range of topics. However, the difference is sufficiently modest that time and budget constraints may justify the use of some video call interviews within a qualitative research study. In-person face-to-face interviewing is often believed to be the ‘gold standard’  in qualiOne alternative to in-person interviewing is video calling. Video calls are an internet based technology which provides the synchronous experience of seeing and hearing the person at the end of the line, allowing for interviews to take place that are effectively face-to-face , arguablVideo calls for qualitative interviewing have drawn consistent criticism for a number of reasons, including technical issues, such as time-lags on video and disconnected calls, –13 the nThe body of literature which compares video calling with in-person interviews is in its infancy. Several works have addressed the use of video calling for qualitative research , 19, 22.There is an established practice of using quantitative measures to assess differences between qualitative interview modes , 24. IrvThis study uses the transcripts from a study which used in-person and video calls with people who have refractory irritable bowel syndrome (IBS) , to idenA convenience sample  of UK reInterviews were conducted either in-person or face-to-face via video call. The decision to use mixed interview modes in this piece of research was taken whilst the study was ongoing and was in response to a sudden recruitment influx from internet advertising (Facebook). It was judged important to capitalise upon this influx rapidly due to the possibility of loss of interest by potential interviewees as the result of the time lag.The transcription started from the point on the interview when the first question was asked by the interviewer. It concluded when the interviewer turned off the recording device, which was done when the answer to the last question was given and the interviewer judged that the interviewee had finished on the topic. Preamble and postamble were unrecorded. The interviewees perceptions of the interview process were not actively sought. Short pauses in speech were not recorded in the transcript, however if a pause was deemed to be unusually long or to denote a higher than average amount of thought an ellipse was inserted. Laughter and audible sighing were recorded with a single word within the transcript but no notes on body language were included.A two-stage process of coding was undertaken. This process started with open coding . Open coThe same topic guide was used for both video call and in-person interviews. The same interviewer (MK) conducted and transcribed all the interviews. The idea to conduct the analysis of the two interview modes did not occur until after the coding had been undertaken. The full protocol of the study is available  as is thSix quantitative measures were used to assess the relative effect upon interviews of the mode of interview, these were: duration of interview in minutes, word count, speech rate, number of codes, number of statements and dominance. These were used to provide quantifiable data over a spread of measures. Two of these measures, duration , 25 and Duration , 25 is aWord count is the total number of words said by both the interviewer and the participant. Word count provides a measure of how much is said in the interview, which may be different from the overall duration of the interview as some people will speak faster and slower or may take longer pauses .Speech rate is a secondary measure calculated by dividing word count by duration to get the average number of words spoken per minute by both interviewer and participant. It is intended to identify whether the use of video calls effect the speed at which people express themselves.A code is the designation applied to any number of comments in a transcript during the analysis phase which are under the same broad topic. There is a tradition of using the number of codes as a quantitative measure within content analysis  but thisThis is the number of statements relating to a code, it is a measure intended to give an idea of the depth and variety captured within the interviews. By using the number of statements as an indicator of how many different ideas or how much additional information was provided on a single code in this by the participants. In practice this means the code ‘ideal therapist’ may encompass multiple statements such as “they’d be able to provide evidence of qualifications” or “someone fairly sort of clean cut”.As the transcriptions were analysed statements were highlighted and either assigned to an existing code or a code was generated for them. This was done within the Nvivo software package so the number of statements was recorded as analysis was conducted. This figure is distinct from the codes as a single code may have multiple statements in support of it e.g. 40 different codes to summarise the topics of 175 separate statements.Broadly this metric can be said to represent the number of distinct comments made on a single topic. However, it is imperfect, there will be some instances where multiple comments on a point have been captured within a single statement as they are delivered within the same brief statement and conversely the same point having been made by the same participant multiple times at some remove from each other and thus have been recorded as multiple statements. The use of a single researcher for coding of the transcripts (MK) who was at the time of coding unaware of the statements ultimate use for this purpose will have meant a continuity of style across both in-person and video call interviews which is likely to standardise the error rate.As statements are being used as a proxy for breadth and variety within the findings it is assumed that there will be only a correlation with the trend, rather than an absolute reflection of it, i.e. higher numbers of statements are likely to suggest more depth and variety but not give a precise indication of how substantial that is, as such small differences in cannot be taken to be meaningful, only large ones. This measure has yet to be validated over multiple studies or in the context of other potential metric or assessments of depth.Dominance is a measure of the percentage of the interview that the interviewer is leading , this is17 people completed an interview. One was removed from this analysis due to being asked an additional question regarding video call hypnotherapy, this being the question which prompted the idea for this analysis it was deemed inappropriate to include it as the interview and coding were conducted with an awareness of how the data may be used for this interview comparison. Additionally, there were questions of how the addition of a question may affect the character of an interview beyond the words directly attributable to that question. This left 16 interviews based on the original topic guide, 8 interviews were in-person, 8 via video call  and a similar breadth of topics (codes). However, in-person interviewees tended to make more individual points (statements) about those topics.Upon examination of the data it becomes apparent that the full transcript word count and the duration of the interview were of minor importance. Equally interviewer dominance, as a percentage difference of the overall length of the interview, is only 3% and tells us little, however it does allow for us to adjust the overall word count to represent the words said by the interviewee alone. This adjusted figure was slightly higher for the in-person interviews over the video interviews 10.1%) which for a sample of this size is arguably negligible. However, the difference in word count observed after interviewer dominance was removed may be explicable by the relative lengths of time since first diagnosis, which is on average 6.4 years higher for the in-person group. The longer a person’s experience of living with IBS the more they are likely to have to say when recounting their experience of being diagnosed, attempting multiple treatments with varying degrees of success, different encounters with and reactions to various clinical situations and how over time it has impacted upon their life. These topics comprised a substantial proportion of the interviews. Either way it should be noted that the ‘Gold standard’ of interviewing (in-person) [0.1% whicWhen examining the quality of those words, arguably the most important point, it was apparent that the number of codes used in the open coding and the amalgamated coding was almost identical. This strongly indicates that both methods produced a comparable breadth of understanding. However, the number of statements on which those codes are founded was quite different, being 23.7 and 19.3% higher for the in-person interviews, open coding and amalgamated coding respectively. This appears to suggest that for these interviewees at least there was a greater spread of distinct opinions, insight and viewpoints expressed within the topics by the in-person group, even if they did not move far from the core point of discussion. The greater number of statements will in some way be related to the higher number of words expressed by the in-person group, but as the number of statements was much higher (23.7 & 19.3%) than the additional number of words (10.1%) exactly what that relationship is remains unclear.People involved in video call interviews used higher rates of speech (speech rate 16.2%). This was possibly due to some heightened anxiety or pressure brought about by the mode of interview. However, all participants in the video call interviews were calling from their home environments which could be considered innately more relaxing than being in a public or an unfamiliar location, particularly true when the sensitive nature of the topic is considered  and poteFrom a qualitative perspective the researchers neither experienced nor noted any consistent difference between the nature and character of the interviews by mode. Even rapport which some have anticipate as being inhibited by the camera  did not As noted, video calls have drawn considerable criticism for technical issues –13 and aIt has been suggested that because video calls provide only a very limited window into an interviewee’s home when compared to a home visit that there may be a loss of contextual understanding of that person’s life by the interviewer . HoweverAs observed by other users of video call interviews they allow for substantial savings in time and cost , 9 and tThe study was conceived after the initial collection and transcription of the data for the original IBS study from which the data was taken, as such researcher bias, something considered a major potential issue in qualitative interviews,  is unlikThere was heterogeneity between the two groups, with the in-person group containing both male participants and all the Black, Asian and Minority ethnic (BAME) participants. The age distribution of the participants is noteworthy with all the older (≥45 years) and younger (≤25 years) interviewees participating in in-person interviews whilst most video call interviews came from those in their 30s and 40s. This could in part be the result of the use of Facebook as part of the recruitment strategy, in the UK Facebook has an average user age of 40,  and all This study found that in-person interviews were slightly superior to video calls in that they produced more words and substantially more statements in support of a similar number of codes. However, the difference was modest, and video call interviews could offer substantial savings of time and budget. As such the use of video call interviews may be justifiable in situations where otherwise the research would not be possible, for example with rare diseases where the population may be highly dispersed or there are situations which are dangerous to enter. In-person interviews should be preferred where older populations are sought due to relatively low levels of familiarity with the technology. Ultimately a mixed mode of interviewing with some interviews being conducted in-person and the costliest in time or money or potentially danger being conducted by video call may be the most efficient balance.


Klebsiella pneumoniae bacteremia is a major cause of morbidity and mortality worldwide. We aimed to compare the clinical characteristics, distribution of capsular types, and antimicrobial resistance of K. pneumoniae bacteremia among community-acquired (CA), healthcare-associated (HCA), and nosocomial infections.K. pneumoniae bacteremia was conducted at Taipei Veterans General Hospital from January to December 2015. Clinical characteristics of K. pneumoniae bacteremia were collected. The K. pneumoniae isolates were subjected to antimicrobial susceptibility testing and capsular genotyping.This retrospective study of patients with K. pneumoniae bacteremia were identified: 70 (20.8%), 102 (30.3%), and 165 (48.9%) presented with CA, HCA, and nosocomial infection, respectively. The 28-day mortality of HCA bacteremia was lower than that of nosocomial bacteremia ; however, that of the HCA and CA bacteremia was similar . CA isolates had the highest prevalence of virulent capsular types (51.4%), followed by HCA (36.3%) and nosocomial isolates (19.4%). The proportion of multidrug-resistant (MDR) isolates was highest in nosocomial infections (41.8%), followed by HCA (23.5%) and CA infections (5.7%).In total, 337 patients with K. pneumoniae are distinct entities, as evidenced by the differences in clinical characteristics, antimicrobial resistance, and capsular types found in this study.CA, HCA and nosocomial  Klebsiella pneumoniae bacteremia is a major cause of morbidity and mortality worldwide .The distribution of capsular types and the presence of K. pneumoniae bacteremia was comparable to that of CA bacteremia but significantly lower than that of nosocomial bacteremia. The rate of MDR phenotype, virulent capsular types and rmpA/rmpA2 genes in the isolates from HCA bacteremia was between those from CA and nosocomial bacteremia. The rate of isolates with virulent capsular types in HCA infection was comparable to that in CA infection. However, the rate of MDR phenotype in HCA infection was higher than that in CA infection. Our findings support that HCA bacteremia is a category distinct from CA and nosocomial bacteremia in terms of the clinical and microbiological features, which was addressed in the previous study [The mortality of HCA us study .K. pneumoniae bacteremia have rarely been compared [K. pneumoniae bacteremia in Canada, 43, 30, and 27% were HCA, CA, and nosocomial infections, respectively [K. pneumoniae isolates with virulent capsular types responsible for liver abscess was more common in East Asia than western countries. Our results underscored the endemic nature of K. pneumoniae liver abscess in Taiwan [The clinical characteristics of CA, HCA, and nosocomial compared . Meathern Taiwan , 21, 22.K. pneumoniae bacteremia had a higher infection-related mortality than CA K. pneumoniae bacteremia  [K. pneumoniae bacteremia was higher than that of CA K. pneumoniae bacteremia  [p = 0.557). HCA bacteremia is rising in prominence and physicians are becoming more familiar with this category, which may contribute to a lower threshold for investigation and detection bias resulting in improved prognosis. Furthermore, the mortality of patients with HCA bacteremia was significantly lower than those with nosocomial bacteremia. In the literature, nosocomial K. pneumoniae bacteremia is usually associated with a higher mortality than community-onset K. pneumoniae bacteremia [K. pneumoniae bacteremia.Our previous study conducted from 2007 to 2010 showed that patients with HCA = 0.001) . HoweverK. pneumoniae have spread to the community or the wild-type strains have evolved and became resistant. The characterization of HCA bacteremia isolates might be helpful in choosing an empirical antimicrobial therapy, as supported by the original intention. Moreover, the surveillance of the resistance pattern of HCA isolates can help in monitoring the development of drug-resistant K. pneumoniae in the community.In the present study, nosocomial isolates are still notorious for the highest rate of resistance (52.7%). The proportion of CA isolates that did not display wild-type susceptibility (14.2%) was similar to that (14.9%) in our previous study conducted from 2007 to 2010 . HoweverK. pneumoniae bacteremia strains [p = 0.02), and the non-typeable strains were more prevalent in nosocomial infections than in community-onset infections  [K. pneumoniae bacteremia via multi-locus sequence typing and pulsed-field gel electrophoresis in Spain [K. pneumoniae isolates had the highest rate of virulent capsular types , followed by the HCA isolates, whereas the nosocomial isolates had the lowest rate of virulent capsular types among the three categories. The distribution of K. pneumoniae isolates with rmpA/rmpA2 genes among CA, HCA, and nosocomial bacteremia was similar to that of the virulent capsular types. Classic K. pneumoniae and hypervirulent K. pneumoniae strains were proposed as the two different pathotypes of K. pneumoniae recently [K. pneumoniae were primarily responsible for community-onset pyogenic infections in Taiwan, and classic K. pneumoniae with antimicrobial resistance usually involved in nosocomial infections. The presence of rmpA/rmpA2 genes and virulent capsular types were usually associated with hypervirulent strains. The current findings support the unique microbiological features between nosocomial versus CA strains. Notably, approximately 19.4% of the nosocomial strains belonged to these virulent capsular types. This is of concern that these virulent strains are spreading and no longer restricted in the community. The virulent strains in hospitals must be monitored to prevent fatal outcomes among susceptible patients. The occurrence of virulent strains in HCA bacteremia might indicate the transmission between community and hospital. The active molecular screening of capsular genotypes and rmpA in K. pneumoniae isolates in hospitals might be an effective strategy in controlling and preventing the spread of infection.Tsay et al. have previously compared the serotypes of community-onset and nosocomial  strains . The ser = 0.01) . Cubero in Spain . Howeverrecently . HyperviK. pneumoniae bacteremia patients. Third, the difference of mortality rates among the three categories might be related to confounding variables that differed significantly and multivariate analysis was not performed to investigate mortality rates further. Fourth, we did not perform multilocus sequence typing of K. pneumoniae isolates to delineate the clonal distribution and genetic diversity among CA, HCA, and nosocomial bacteremia. Finally, our study was conducted in a single tertiary-care teaching hospital where the patient populations, clinical practice, and treatment outcome may differ from those in other non-tertiary-care hospitals. Thus, the results may not be generalizable.The present study had several limitations. First, incomplete medical records excluded a certain proportion of the total population that should have been included. However, only 2 patients were excluded accounting for very low proportion (0.6%) of the total population. Second, the absence of evidence pertaining to prior antibiotic exposure does not equate to evidence of absence, especially in the CA and HCA K. pneumoniae bacteremia is a category distinct from CA and nosocomial infections in terms of clinical and microbiological features. The characterization of the clinical characteristics of CA, HCA, and nosocomial bacteremia will help professionals to better manage patients. Further studies on the microbiological characteristics of HCA strains must be conducted to accurately identify the transmission of virulent or antimicrobial-resistant strains between community and hospital. We also encourage further studies from different geographical locations to assess the global distribution and trends in this research topic.HCA


However, millimeter wave (mmWave) communications in the 60 GHz band has gained considerable interest from the industry and academia because it promises gigabit wireless connectivity in the huge unlicensed bandwidth . This massive unlicensed bandwidth offers great potential for addressing the demand for 360-degree video streaming. This paper investigates the problem of 360-degree video streaming for mobile VR using the SHVC, the scalable of High-Efficiency Video Coding (HEVC) standard and PC offloading over 60 GHz networks. We present a conceptual architecture based on advanced tiled-SHVC and mmWave communications. This architecture comprises two main parts. (1) Tile-based SHVC for 360-degree video streaming and optimizing parallel decoding. (2) Personal Computer (PC) offloading mechanism for transmitting uncompressed video (viewport only). The experimental results show that our tiled extractor method reduces the bandwidth required for 360-degree video streaming by more than 47% and the tile partitioning mechanism was improved by up to 25% in terms of the decoding time. The PC offloading mechanism was also successful in offloading 360-degree decoded (or viewport only) video to mobile devices using mmWave communication and the proposed transmission schemes. Recently, 360-degree video streaming for virtual reality (VR) that is currently available on some major video platforms, such as YouTube, Facebook, etc. has emerged. However, the computing power of mobile devices and bandwidth of 2.4 GHz or 5 GHz wireless networks are limited compared to the requirements of high-quality VR. Let us provide some numbers to illustrate this problem. The VR viewport is defined by a device-specific viewing angle  that delimits horizontally the scene from head direction center, called the viewport center. To ensure good immersion, the pixel resolution of the displayed viewport is high, typically 4K  that may have dependencies on the CTUs of other tiles. The VR viewport presents a portion of 360-degree video, namely field of view (FOV) [thors of  proposedThe moving picture experts group (MPEG) standard leverages motion-constrained tile sets (MCTS) as a classic way of limiting MVs in the current picture . MCTS liPixels/degree: 40 (number of pixels per degree).Resolution: 11,520 Frame rate: 90 fps (90 fps can prevent nausea through low delay).Environment-based or scene-based audio (360-degree surround sound and object-oriented audio).Maximum video and audio delay: 20 ms (time between user interaction and VR image and audio).According to 116th MPEG meeting, the following requirements should be met to support high-quality VR video streaming [To satisfy the above requirements, a data rate from0.9546 Gbps to 19.11 Gbps should be supported . Among eAs summarized in The goal of proposed architecture is to provide the video of highest quality in limited mobile VR environments, as shown in The functionality of the proposed tiled-based method is described in Proposed Step 1: The motion vector of undecoded tile at EL is replaced by the upsampled BL.In step 1, we suggest the use of the upsampled PUs at the BL to overcome the problem mentioned in Proposed Step 2: Available tile encoding in EL using upsampled BL and decoded tile of EL.Step 1 solves the problem of referring to the outer region of the viewport. However, as step 1 uses only an upsampled PU at the BL as a reference list, that PUis still available for ILP. Therefore, as shown in The SHVC performs TIP within the same layer and inter layer prediction (ILP) between different layers through an up-sampling filter . This woDifferent from SHVC, the HEVC  encoder eight-tap filter to interpolate luma prediction. When the eight-tap filter is applied horizontally, three pixels to the left and four pixels to the right of the current pixel are used. When applied vertically, the top three pixels and the bottom four pixels from the current pixel are used. x and y pixel values at the top and left of the current PU can be obtained using the getCUPelX and getCUPelY functions, while the x and y pixel values at the bottom and right can be obtained by adding the values obtained from the getWidth and getHeight functions in the HM and SHM software. However, if the current PU is not in the 2N × 2N mode, its position should be changed. Because the four functions discussed above return a value based on the 2N × 2N mode, the position and size of the PU can be obtained by considering the position of the PU in eight partition modes.When the reference pictures have already decoded, the tiles are not temporally independent even the tiles are spatially independent to support parallel processing . TherefoH candidate block is selected, independent tile transmission is not guaranteed because it uses motion information from another tile. The modified HM and SHM first determines whether the current CTU is located on the right side of the tile, that is obtained using the getRightEdgePosInCtus function. The current CTU position is obtained using the getFrameWidthInCtus and the getCtuRsAddr functions. Based on whether the current PU in the CTU is on the right side of the CTU, the position of the current PU is obtained using deriveRightBottomIdx. The getNumPartInCtuWidth and the getNumPartInCtuHeight functions are used to determine if the current PU position is on the right side of the CTU. If both conditions are met, the H block is excluded from the candidate.The HEVC and SHVC encoders use advanced MV prediction (AMVP) and merge to reduce the amount of motion information in the inter prediction. Both modes use spatial and temporal candidate blocks. As mentioned in PeopleOnStreet that have a resolution of Res is the resolution of the tile, Comp is the decoding complexity of the tile. The computed complexity Comp is given by ) and implicit others . As discFinally, if we obtain the regression model, the performance ratio between big and little cores, and the number of big and little cores, we can calculate sL as in  and 8)..ResB andsL as in  and (8),To solve the high resolution of 360-degree viewport streaming. Instead of other wireless 802.11 technologies, the mmWave link is applied to support high speed.To avoid the overflow issue or quite low performance issue of high-resolution video processing in mobile devices in terms of decoding rate , the decAs mentioned in In MVP, the viewport is extracted and encoded at the EL. To overcome the limitations in power and computation for handling the viewport at high resolution, MVP uses PC offloading to decode the viewport and transmit the decoded viewport to mobile devices using mmWave connections. The main idea behind the proposed scheme is as follows:The first synchronization mechanism is implemented as a classic method of creating a reliable session by sending ACK messages and lost packets on mmWave channels as shown in In this section, the proposed method is implemented and evaluated through several unit tests.This experiment uses test sequences selected by the Joint Video Expert Team (JVET), as shown in PeopleOnStreet  has four big and four little cores, but this experiment considers only two big and four little cores because two big cores are always on online state while other two big cores are on offline state usually for power saving. In this work, we used the HM15.0 encoder and two 4K test sequences  and a 0.05 dB PSNR over the original encoding because the MV and temporal candidates of the AMVP and merge were limited. However, SHM and HM saved an average of 48% and 47% bitrates when transmitting four ROI tiles among nine other tiles. In addition, SHM and HM saved at least 49% up to 87% of the bitrate with four sequence averages when only one tile was sent. Moreover, we presented a novel tile partitioning method for parallel decoding on asymmetric multicores. The size of the non-uniform tiles is based on the regression model of the computational complexity per video resolution. This contributed to a 25% speeding up of the decoding process. This paper also proposed PC offloading mechanisms using mmWave communication. allowing PC decoding full video (or viewport only) and raw data forwarding to mobile devices over the mmWave connections. Two synchronization schemes were applied to reduce the effect of packet loss and fluctuating mmWave channels. In the future, we will continue to develop the concept architecture by merging the currently achievable results and considering relay prototypes to support blockage cases for mmWave communications.References yes