Repetitive sequences represent about half of the human genome. They are actively transcribed and play a role during development and in epigenetic regulation. The altered activity of repetitive sequences can lead to genomic instability and they can contribute to the establishment or the progression of degenerative diseases and cancer transformation. In this work, we analyzed the expression profiles of DNA repetitive sequences in the breast cancer specimens of the HMUCC cohort. Satellite expression is generally upregulated in breast cancers, with specific families upregulated per histotype: in HER2-enriched cancers, they are the human satellite II (HSATII), in luminal A and B, they are part of the ALR family and in triple-negative, they are part of SAR and GSAT families, together with a perturbation in the transcription from endogenous retroviruses and their LTR sequences. We report that the background expression of repetitive sequences in healthy tissues of cancer patients differs from the tissues of non-cancerous controls. To conclude, peculiar patterns of expression of repetitive sequences are reported in each specimen, especially in the case of transcripts arising from satellite repeats. Breast cancer is still the leading cause of mortality among the female population in developed countries. In post-menopausal women, it accounts for 23% of all cancer deaths . Breast The mammalian genome is pervasively transcribed and only a small portion of the transcriptional output has protein-coding potential . The nonA multifaceted category of ncRNAs, of growing interest due to their roles in human health and diseases, is represented by the transcripts arising from repetitive DNA sequences (RS), i.e., DNA sequences that are present in multiple copies in the genomes, with low or nonexistent coding potential. RS represent about 45% of the human genome and are differentially transcribed in many tissues . In mammIncreased levels of heterochromatic repetitive satellite-coded RNAs in mammary glands induce breast tumor formation in mice, altering the BRCA1-associated protein networks that are required for the proper stabilization of DNA replication forks that in turn lead to genomic instability . In humaIt is known that LINE-1-encoded retrotranscription activity is widespread and its inhibition can reduce the rate of proliferation and promote the differentiation of breast cancer cells . LINE-1 BRCA1 and BRCA2 genes essential to genomic rearrangements or genetic mutations leading to etiopathogenic, prognostic or predisposing mutations of breast cancers, both in somatic and germ lines [There are many reports regarding Alu and other SINE elements within or surrounding rm lines ,49,50,51rm lines , suggestThere is significant evidence regarding the use of HERV-K-coded proteins as tumor markers and immunologic targets ,56,57,58BRCA1 mutation in three unrelated French breast/ovarian cancer families, that can be generated by an abortive integration of the human Tigger1 DNA transposon, has been postulated [DNA transposons are the less active and less well-studied class of RS in humans. Nevertheless, few reports suggest their role in breast cancer ; howeverstulated .The main aim of this work was to analyze the transcripts arising from the repetitive sequences in a cohort of breast cancer specimens. Overall, we report peculiar patterns of expression and a diffuse upregulation of satellite transcription specific for each histotype.Bacillus subtilis SMY strain) have previously been described [The method of analysis and positive and negative controls   using thp-value correction for multiple comparisons was performed with the Benjamini and Hochberg method [The differential expression analysis was performed with the DESeq2  algorithg method .We analyzed the data published in the European Nucleotide Archive (ENA), RRID:SCR_006515, study accession: PRJNA292118 ,5. The dThe expression of repetitive sequences has been retrieved and analyzed accordingly to the previously described pipeline  with minA comparison between the 15 invasive breast cancer specimens and 18 controls indicated a panel of RS differentially expressed in the two conditions . ALR, BSIn ‘HER2-enriched’ breast cancer, an upregulation of HSATII satellite expression is reported . RegardiA detailed analysis of the comparison between the expression of ANT and the normal controls is reported in higher) or decreased (GGAATlower).We also report that the vast majority of the specimens analyzed showed an altered expression of GGAAT repeats in comparison with their specific ANT, either increased  or decreased (GGAATlower). The GGAAThigher specimens have a higher ki67 staining [higher.We also report that the vast majority of the specimens analyzed showed an altered expression of GGAAT repeats in comparison with their specific ANT, either increased , which ultimately need to be investigated further.The analysis of transcripts arising from RS in breast cancer specimens classified following the current molecular markers showed a great interspecimen variability, with peculiar patterns of altered RS transcription. Overall, there is an evident alteration in the transcripts arising from satellite repeats and, in specific cases, from SINE and endogenous retrovirus sequences. The expression from healthy adjacent tissues of cancer specimens showed an altered expression of RS transcription when compared to the transcription of healthy donors. If the data presented here are confirmed and extended in a larger population, RS expression may play a role in the molecular classification and stratification of patients or even be potentially adopted as a biomarker in liquid biopsy.


Amycolatopsis eburnea lipases based on the SASA patterns. This information could help in recognizing the structural stability and conformation as well as precise clustering them for revealing lipase evolution.Solvent-Accessible Surface Area (SASA) as the one dimensional structure property of the protein considers as the measuring the exposure of an amino acid residue to the solvent in one protein. It is an important structural property as the active sites of proteins are mostly located on the protein surfaces. The aim of this paper is to provide the clear information on different Amycolatopsis eburnea lipases to exploit the enzyme stability and evolution. Amycolatopsis eburnea lipase sequences were retrieved from biological database. Six structural conserved regions and their residues were identified. Total Solvent Accessible Surface Area (SASA) and structural conserved-SASA with unsupervised agglomerative hierarchical algorithm were clustered lipases in three distinct groups (99/96%). The minimum SASA of nucleus residues was related to Lipase-4. It is clearly shown that the overall side chain of SASA was higher than the backbone in all enzymes. The SASA pattern of conserved regions clearly showed the evolutionary conservation areas that stabilized Amycolatopsis eburnea lipase structures. This research can bring new insight in protein design based on structurally conserved SASA in lipases with the help of a machine learning approach.The wealth of biological databases provides a valuable asset to understand evolution at a molecular level. This research presents the machine learning approach, an unsupervised agglomerative hierarchical clustering analysis of invariant solvent accessible surface areas and conserved structural features of  Amycolatopsis eburnea lipases and clustering of conserved structural-SASA with the help of the unsupervised agglomerative hierarchical method (machine learning approach) toward identification of hot spot structures for protein stability and conformation. The results will help in the design and engineering of new enzymes.Hydrophobic forces in proteins play a vital role in the stability, folding and protein–protein interaction ,2. The rGlycerol ester hydrolase or triacylglycerol acylhydrolase is (E.C.3.1.1.3), a fat splitting enzyme which is also called Lipase . The proAmycolatopsis genus [Amycolatopsis species require study to find the details of structure and diversity of important enzymes such as lipases [Amycolatopsis eburnea, one of the species in the Amycolatopsis genus, has a symbiotic relationship with mycorrhizal fungi; however, the details of this mechanism need to be investigated.It is clearly established that around seventy medicinal and agricultural microbial products are from the Actinobacteria phylum. The famous ones are different antibiotics produced by the is genus ,21. Thisis genus ,23,24,25 lipases ,28,29,30Computational characterizations of enzymes with the help of machine learning algorithms offer a great opportunity to speed up the systematic classifications ,32. TheyThe lipases with microbial origin can be isolated from different cellular compartments, either extracellular, peripheral protein or intracellular enzymes ,35,36. IAmycolatopsis eburnea with their inevitable place in industry is noticeable. Therefore, the aim of this research paper is to provide insight and clear information on different Amycolatopsis eburnea lipases based on the SASA. This information can help in recognizing the lipases structural stability and conformation. The clear information of each amino acid in the structure could help in designing the new lipase enzyme with better functionality. Furthermore, this precise clustering of specific amino acids can demystify the lipase evolution and even enzyme functionality.The lack of information on structural stability of lipase from the Amycolatopsis eburnea lipases. The physiochemical parameters and 3-D homology models were calculated with the help of bioinformatics-server (https://www.expasy.org/) (accessed on 1 February 2020) [The sequence data were retrieved from the National Center for Biotechnology Information (NCBI) database for ry 2020) . Furtherry 2020) . The phyry 2020) . The secry 2020) . The cluThe solvent accessible surface area of each enzyme was calculated according to Fraczkiewicz and Braun . The CarAmycolatopsis eburnea lipases. The cluster analyses of SASA patterns of lipases were performed with unsupervised agglomerative hierarchical clustering method as a machine learning approach with the help of python 3.9 programming language (http://www.python.org) (accessed on 21 August 2021).Structurally conserved regions (SCR) for each enzyme were identified with the help of Chimera with defaults parameters  and solvAmycolatopsis eburnea lipases showed in Physiochemical features of Amycolatopsis eburnea lipase sequences were again modeled with deep learning de novo modeling as described by Yang and coworkers [The three-dimensional structure of the enzymes was modeled with homology modeling with the help of Swiss institute of bioinformatics-server . All modoworkers . The resAmycolatopsis eburnea lipases showed less frequency of His, Met and Cys and Trp compared to other residues  in each enzyme is shown in Amycolatopsis eburnea lipases is shown in Hierarchical clustering of structurally conserved regions-SASA of Amycolatopsis eburnea lipases. The conserved regions SASA might indicate the minimum SASA which was essential for stability of the protein and folding.The structurally conserved regions  showed tAmycolatopsis eburnea lipases. It was observed that GLY and Val are the most frequent residues in conserved regions with 28 and 27 repeats. Different residues were shown in Amycolatopsis eburnea lipases.The similarity of SASA in conserved regions could shed light on the conserved and preferences of residues for the stability of There is an increased concern for lipase as the third most important enzyme in the market for hydrolyzing triglycerides in different media ,46,47,48The large applications of Lipases in many fields from food to medicine are due to their functionality to work in different media; these diverse applications are the reason for a huge demand in the market .Amycolatopsis eburnea) provided a noble prospect to work on for understanding the lipase production. This genus (Amycolatopsis) of bacteria showed many antibiotic productions in different conditions [Different sources of lipases were reported in the past; however, the bacterial sources are more suitable and get the better chance for industrial applications . Applicanditions ,56. Thernditions .Amycolatopsis eburnea can provide clearer information for lipase classification and even help to introduce more clear understanding of lipase evolution. As lipases are water soluble and their substrates are mostly insoluble, their structures should dictate the specific functional activity [Recently published genetic diversity of lipase in bacteria showed great differences in lipase characterizations. However, they revealed a conserved sequence which contained penta-peptide (Gly-X-Ser-X-Gly) . Seven gactivity . Functioactivity .We should also mention that lipases are substrate specific, as well as structurally chemo-, region- and stereo- specific. Three most recognized groups of lipases can mention here as non-specific lipases, 1, 3-specific lipases and acid-specific lipases based on their catalyzing activity on triglycerides substrate in different systems. The preferred lipases for industry should have a low reaction time and remain resistant to various pH beside the activity in non-aqueous media . The priAmycolatopsis eburnea lipases. This structural conservation contained GLY and Val with higher percentage residues that imposed the stability and folding functionality to Amycolatopsis eburnea lipases. SASA features of these regions also deduced the hydrophobic contact information from the hydrophobic residues in lipase structures. The secondary structure length and loops in Amycolatopsis eburnea lipases were exactly related and substantially conserved. The conserved SASA could be a result of selective pressure on molecular conservation. Here, we identified 8 clusters, of which their mean of the SASA were very close. These 6 conserved regions were great tools for describing the stability and surface plasticity of lipases in different environments and their substrate specificity [In our research, the role of interacting hydrophobic residues in conserved structural regions was clearly presented. The lipases presented here clearly showed that they are homologous and the structural homology features are recognizable based on their similarity and phylogenetic dendongram. However, the need for finding structural similarity was necessary to establish a common ancestry. This structural conservation presented here clearly showed the surface plasticity in cificity ,62,63.The finding of the role of these residues in folding or function could even be clearly answered as most of the amino acids in conserved regions are considered hydrophobic residues to some extent. Therefore, they would act in stability and folding conservation of lipases. This research could support the results and hypotheses in finding the specific residues to develop the better enzyme with mutation approaches. Furthermore, it clearly could help in sequence- structure correlation, role of individual residues in folding, stability and function.Amycolatopsis eburnea lipases. This pattern of SASA/hydrophobic positions was observed and clearly conserved. The results showed the compensating amino acids residues that might occur during evolution were conserved in SASA features. It should be noted that amino acids mutation could be detected in conserved regions also. Thus, the only conserved feature in the conserved structure was SASA and hydrophobic features. Then the results showed the specific SASA conservation pattern to impose the native folding in homologous lipases from Amycolatopsis eburnea. Therefore, the significant correlation between sequences, conserved structural regions and SASA features was observed. This information could extend lipase structural information and describe the algorithms to predict SASA protein contact map in future.It is important to mention here that the results clearly showed that there were specific structural constraints with specific residues SASA features conserved in Microbial lipase production needs to introduce better microbes and optimize more suitable environmental conditions. The lipase structure identification is the essential part of the system to find the better source of microbe for their production. Therefore, microbial sources can play the vital role in this selection. Traditionally many microbial sources selected for industrial production based on their amount of lipase production. However, the functionality of lipases and their efficiency can improve significantly by finding the better structure ,50,64.Amycolatopsis eburnea for industry and evolution purpose is inevitable [These days with the help of huge bioinformatics data in protein databases and computational analysis, finding the better lipase structure for industry is more feasible. On the other hand, as the structure of enzyme was proved to be species specific therefore working on lipases from specific species is more reasonable and practical. It should be mentioned here that working on lipase structural analysis would be a great help in finding the evolution of enzyme specially to find the essential residues to track the homology relation. On the other hand, different environments could play substantial roles in functionality of lipases. Soil usually provides the vital habitat for lipase microbe alone and in interaction with plants and other biofilm. Thus, research on lipases with soil microbe origin such as evitable .Amycolatopsis eburnea had specific conserved hydrophobic contact area. This feature robustly categorized the lipases in two clusters. There was another report of lipases categorizing with other features that found seven groups, however the SASA feature didn’t considered for categorizing [Bacterial lipases with soil origin had shown huge diversity and variation in molecular and biochemical characterization. However, the conserved structural area such as residues related to active site (serine residue enclosed with conserved penta-peptide (Gly-X-Ser-X-Gly)) was conserved in all lipases . In ordegorizing .Furthermore, designing the new lipase as well as new primer and probe could get great help from finding the conserved SASA feature ,67. PuriBacillus stratosphericus) to 70 kDa [Pseudomonas gessardii) to 10.8 (Enterococcus faecium) [Acinetobacter sp. XMZ  to 80 °C (Janibacter sp. R02) [Amycolatopsis eburnea lipases was diverse and from 24 kDa to 44 kDa however the average conserved SASA area of lipases is around 43 Å2.Lipase showed the molecular mass from 19 (o 70 kDa  with actsp. R02) . Our resAmycolatopsis eburnea lipases can apply as the great asset for precise engineering of lipases for agricultural and industrial purposes.It is important to mention that all modification of lipases from chemical modification to immobilization and UV and gamma ray irradiations as well as amino acid modification and mutagenesis need great investigate to find the effect of them on conserved SASA of lipases ,73. The The 3-D structures of lipases with ɑ/β-hydrolase fold architecture provided here can be an outstanding tool for protein modeling and engineering the lab experiments ,75. The Amycolatopsis eburnea with great impact on agricultural and industrial sectors have specific structural patterns. Therefore, for developing and designing the new lipases the substantial insight on Amycolatopsis eburnea lipase structure, hotspots were presented with a machine learning approach. Structural landscapes of lipases with specific conserved SASA features from Amycolatopsis eburnea showed the better potential to be the model to design and develop synthetic lipases with an unsupervised agglomerative hierarchical method. Finding the conserved SASA of Amycolatopsis eburnea lipases showed a clear need for having the specific residues with specific SASA be in the structure/sequence of the enzyme for its stability and conformation. This pattern in the enzyme structure can help in the design of the synthetic lipase and even provide a great asset to find the homology of this enzyme from an evolutionary point of view. Amycolatopsis bacteria with symbiotic relationship with mycorrhiza can even be good examples for soil-, bacteria- and fungi-plant interactions research, and the SASA patterns in the structure of lipase enzymes can help to investigate and understand this symbiosis in future research.It is clearly shown that lipases from


The structure and function of epithelial cells are critical for the construction and maintenance of intact epithelial surfaces throughout the body. Beyond the mechanical barrier functions, epithelial cells have been identified as active participants in providing warning signals to the host immune and inflammatory cells and in communicating various detailed information on the noxious challenge to help drive specificity in the characteristics of the host response related to health or pathologic inflammation. Rhesus monkeys were used in these studies to evaluate the gingival transcriptome for naturally occurring disease samples (GeneChip® Rhesus Macaque Genome Array) or for ligature-induced disease (GeneChip® Rhesus Gene 1.0 ST Array) to explore up to 452 annotated genes related to epithelial cell structure and functions. Animals were distributed by age into four groups: ≤ 3 years (young), 3–7 years (adolescent), 12–16 years (adult), and 18–23 years (aged). For naturally occurring disease, adult and aged periodontitis animals were used, which comprised 34 animals . Groups of nine animals in similar age groups were included in a ligature-induced periodontitis experiment. A buccal gingival sample from either healthy or periodontitis-affected tissues were collected, and microarray analysis performed. The overall results of this investigation suggested a substantial alteration in epithelial cell functions that occurs rapidly with disease initiation. Many of these changes were prolonged throughout disease progression and generally reflect a disruption of normal cellular functions that would presage the resulting tissue destruction and clinical disease measures. Finally, clinical resolution may not signify biological resolution and represent a continued risk for disease that may require considerations for additional biologically specific interventions to best manage further disease. The structure and function of epithelial cells are critical for the integrity of epithelial surfaces throughout the body. These structures limit the access of pathogens that interact with mucosal surfaces to invade and initiate disease . Beyond The signals that elicit epithelial responses include microbial-associated molecular patterns (MAMPs) that trigger inflammatory and antimicrobial peptide responses and the release of danger-associated molecular patterns (DAMPs) –9 to comAccumulation of microbial biofilms supra- and sub-gingivally trigger gingival tissue reactions, leading to biologic and clinical responses described as gingivitis –15. PersWe have previously described the use of the non-human primate model of naturally occurring and ligature-induced periodontitis to identify gingival tissue transcriptome patterns and to examine an array of biologic pathways that are altered by disease and affected by aging –30. ReceAs epithelial cell functions and an intact epithelium are critical to periodontal health, we extended this non-human primate model to pattern the transcriptome of gingival tissues with naturally occurring and ligature-induced periodontitis with aging as a co-factor for gene expression profiles.Macaca mulatta) housed at the Caribbean Primate Research Center (CPRC) at Sabana Seca, Puerto Rico were used in these studies. As reported previously, the non-human primates are fed a 20% protein, 5% fat, and 10% fiber commercial monkey diet . The diet is supplemented with fruits and vegetables, and water is provided ad libitum in an enclosed corral setting. A protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Puerto Rico, allowing clinical measures of the periodontium, including probing pocket depth (PPD), bleeding on probing , as we have described previously, and the collection of the gingival tissue biopsies  with x gene expression value, μ means across the samples, and σ standard deviation across all samples.The expression intensities across the samples were estimated using the Robust Multi-array Average (RMA) algorithm with probe-level quintile normalization, as implemented in the Partek Genomics Suite software version 6.6 . The differential expression was initially compared using one way ANOVA across time points within an age group. For genes that had significant mean differences, two sample p < 0.01 and/or a change of >1.5/<−1.5 fold compared to baseline values.The means of transcript data across all monkeys in the same age and disease category were calculated to yield 20 monkey age-disease groups. Gene expression data of 429 genes and 20 monkey age-disease groups was normalized on a scale of 0.130–0.297, with the lowest value set at 0.130 and the highest value at 0.297 to generate the heatmap and dendrograms for the cluster analyses . The unrooted dendrogram was constructed. The fan dendrogram was constructed by calculating the distance matrix with the Euclidean method, followed by Ward 2 linkage clustering method using the Ape package in R version 5.5, depicting the transcript data of 164 genes that were determined from univariate analysis to be significant at p ≤ 0.01 and/or increased/decreased by 2-fold or greater in the healthy aged tissues. The plot for the naturally occurring periodontitis tissues summarizes the gene expression in adult and aged diseased tissues compared to healthy adult samples. In these analyses, 39 and 19 genes were significantly different in the aged and adult disease samples, respectively. Furthermore, 31 (aged) and 26 (adult) genes were expressed at a 2-fold or greater difference in the disease samples.Data derived from the groups of healthy adult and aged animals and the same age groups with naturally occurring periodontitis are presented in However, within this large assortment of epithelial cell-related genes, there was a more limited group of the genes that demonstrated differential expression in naturally occurring periodontitis. With ECM structural genes, the majority of genes were decreased with disease , while Ep < 0.01) differentially expressed at initiation (2 weeks), with somewhat fewer expressed during early (1 month) and late (3 month) disease progression. This was also emphasized with a greater number of genes showing a fold-difference compared to the samples from progressing disease. A similar pattern was noted in the adult and aged animals in relation to changes occurring with disease initiation. However, there was a predilection for an increased frequency of changes in the adult animals at 1 month, while this magnitude of significant differences was primarily noted in the aged animals at the 3 month time point. A benefit of the use of the non-human primate to model the immunobiology of periodontitis is the capacity to integrate a ligature-induced disease process into the experimental design. In this model, it allows a specific identification of the initiation, progression, and resolution of the disease lesions. ng genes  were eleng genes  includedng genes  showed dng genes  in all ang genes  were evang genes  were domng genes  expressiWithin the larger array of various functional categories, beyond just the magnitude of expression, it was useful to highlight those genes related to epithelial cell functions that demonstrated a larger fold-change with disease and resolution comparted to heathy tissues . The genThe integrity of the epithelium and broader functions of the epithelial cells are critical for maintaining or returning to homeostasis. Thus, we examined the expression of this array of genes related to the clinical parameters of disease, e.g., bleeding on probing and probing pocket depth, in the adult and aged animals, reflecting the patterns that might be expected in chronic periodontitis in humans. p < 0.01 and/or differed from baseline health by >1.5-fold at any of the time points. The results refined the cluster analytics and identified 18 clusters that discriminated the samples based on disease and age. These relationships are also demarcated in a fan graphic analysis . This anThe data were then subjected to a pathway analysis to highlight those pathways primarily represented by the altered expression of this gene array related to epithelial cell biology. An intact epithelial barrier is a critical component of the oral mucosa and clearly interfaces with the complex oral microbiome to maintain homeostasis. Additionally, beyond this mechanical barrier, considerable evidence has been generated regarding critical innate immune functions of the epithelial cells in production of protective factors and communication activities with resident and emigrating inflammatory and immune cells in health and disease. However, how these cells or tissues function at the molecular level across their portfolio of biological activities during the transition from health to inflammation and the formation of a periodontal lesion is not clear. Studies in human disease generally compare the features of clinically healthy gingival tissues with samples from sites with measurable clinical features of bleeding, pocketing, and attachment loss. However, a critical assessment of the characteristics and kinetics of the changes that occur with disease initiation and progression is limited in the human model. This occurs since the clinical features that can be measured are not necessarily coincident with the biological changes that fundamentally underpin dysbiotic microbiome changes and dysregulated host responses . Cell biThis study examined the biology of epithelial cell related gene expression in gingival tissues from non-human primates with naturally occurring and experimental ligature-induced disease. We also detailed the impact of age on the features of these responses and related those to the clinical disease features in the animals. Specifically, we quantified the expression of 452 genes that are related to epithelial cell biological functions. These included those reflecting extracellular matrix structure, ECM remodeling, cellular adhesion, cytoskeleton regulation, junction associated, growth factors, cell surface receptors, kinases, and response markers of inflammation and antimicrobial peptides.Across the functional categories of the epithelial related genes were substantial similarities in the expression of these individual genes irrespective of the age of the animals. Generally, ECM structural genes and junction-associated genes were decreased in disease, and these changes were noted with initiation of the disease lesion. In contrast, genes representing cytoskeleton regulation, cell adhesion, cell surface receptors, and ECM remodeling were all increased with the disease process. Growth factor genes appeared altered in expression related to age of the animals, even in samples from healthy sites. Related to intracellular biological activities, the array of kinases was generally decreased in disease, while transcription factors appeared to be predominantly increased in the disease tissues. The summation of these gene expression differences provides a snapshot of the biology of gingival tissues that are particularly related to altered functions of the epithelium and epithelial cells, which clearly occur in the disease lesions, but are seemingly less affected by age.Fusobacterium nucleatum [Various reports have attempted to examine transcriptomes in complex tissue environments and in cell cultures to identify “hub genes” for epithelial cells functions. The hub genes are described as genes with high connectivity in tissues and diseases. Reports using Weighted Gene Co-expression Network Analysis (WGCNA) , 54 deteucleatum . Of inteAnother important question in periodontal biology is related to providing a clearer understanding of the temporal nature of changes in responses reflecting a breakdown in the integrity of the tissues that would be a harbinger of clinical disease outcomes. We have reported on the kinetics of the biology of the lesion using this non-human primate model . SpecifiFinally, this disease model enables us to explore the patterns of responses in gingival tissues where the periodontal lesion has been clinically resolved. Thus, we addressed the question of whether the epithelial cell biology of the resolved sites reflected the transcriptome patterns of healthy non-diseased gingival tissues. Across all age groups, there were numerous differences in the epithelial genes in the resolution samples, almost exclusively with decreased expression compared to baseline healthy samples. In this regard, the expression of these genes most often paralleled the expression in diseased tissues and was most prevalent in ECM structural, ECM remodeling, and cell adhesion genes. These results are consistent with clinical findings that suggest that the greatest risk for a periodontal lesion is in tooth sites with a previous episode of destructive disease , 15. ThiThe overall results of this investigation suggest a substantial alteration in epithelial cell functions that occurs rapidly with disease initiation. Many of these changes are prolonged throughout disease progression and generally reflect a disruption of normal cellular functions that would presage the resulting tissue destruction and clinical disease measures. Additionally, the overall profile of gene expression of epithelium-related genes differed in younger and older animal samples. There is an elevated prevalence and severity of periodontitis with aging suggesting a potential role for epithelial cell biology with increased age. Finally, clinical resolution may not signify biological resolution and continued risk for disease that may require considerations for additional biologic specific interventions. Future transcriptomic studies evaluating specifically dissected gingival epithelial tissue would be needed to confirm some of the main findings described in this report.https://www.ebi.ac.uk/metagenomics/, E-MTAB-1977; https://www.ncbi.nlm.nih.gov/geo/, GSE180588.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found at: The animal study was reviewed and approved by a protocol that was approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Puerto Rico.OG and JE were responsible for the design, conduct of the experiment, clinical data and sample collection, interpretation of the data, and preparation of the manuscript. SK was responsible for the preparation of the tissue specimens for analysis and the microbiome samples. LN provided advanced statistical analysis and contribution to the methods. LO, MN, and JG-M all provided support for the implementation of the non-human primate disease model, data collection, and review of the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by National Institute of Health grant P20GM103538. We express our gratitude to the Caribbean Primate Research Center (CPRC) supported by grant P40RR03640, specifically by Dr. Armando Burgos and the Center for Oral Health Research in the College of Dentistry at the University of Kentucky.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


A 53-year-old woman was brought to the emergency room with headache and progressive deterioration of consciousness. Radiological examinations revealed acute subdural hematoma extending along the cerebellar tentorium to the falx cerebri, and a mass lesion with hemorrhage in the left cerebellum, with acute hydrocephalus. Emergency tumor and hematoma removal with decompressive craniectomy of the occiput was performed. Histopathological diagnosis was microcystic meningioma. Postoperatively, the patient recovered to clear consciousness with sequelae of left cerebellar ataxia, cerebellar dysarthria, and vertigo. This case of tentorial microcystic meningioma associated with acute subdural hematoma in the posterior cranial fossa is extremely rare, with only reported 4 similar cases. Meningioma is one of the most common benign brain tumors and is characterized by abundant vascularity A 53-year-old woman with no previous medical history was brought to the emergency room with headache and progressive deterioration of consciousness. On arrival, her consciousness was lethargic . Her pupils were isocoric, but the left eye was adducted, which indicated left abducens nerve paralysis. No paralysis of the extremities was observed. Head computed tomography revealed acute subdural hematoma extending along the cerebellar tentorium to the falx cerebri and a hemorrhagic mass lesion of 4 cm in maximum diameter in the left cerebellar hemisphere. The cerebral aqueduct was deviated to the right, resulting in acute hydrocephalus . PrecontEmergency tumor and hematoma removal with decompressive craniectomy of the occiput was performed. The tumor was extramedullary and attached to the tentorium. The surgery was completed leaving only slight residual supratentorial lesion . PostopeIntracranial hemorrhage due to brain tumor accounts for 0.9%-10.2% of all cases of intracranial hemorrhage Intracranial hemorrhage with meningioma has various causes, such as 1) hemorrhage due to failure of the feeding blood vessels due to hypertrophy and dilation of the blood vessel walls as the tumor grows, 2) hemorrhage due to failure of abnormal blood vessels in the tumor, 3) hemorrhage due to compression and occlusion of the veins and venous sinuses around the tumor as the tumor grows, and 4) hemorrhage due to trauma We describe an unprecedented case of tentorial microcystic meningioma associated with acute subdural hematoma of the posterior cranial fossa. The preoperative differential diagnosis was difficult because of the atypical preoperative imaging findings of the meningioma.


The development of smart applications has benefited greatly from the expansion of wireless technologies. A range of tasks are performed, and end devices are made capable of communicating with one another with the support of artificial intelligence technology. The Internet of Things (IoT) increases the efficiency of communication networks due to its low costs and simple management. However, it has been demonstrated that many systems still need an intelligent strategy for green computing. Establishing reliable connectivity in Green-IoT (G-IoT) networks is another key research challenge. With the integration of edge computing, this study provides a Sustainable Data-driven Secured optimization model (SDS-GIoT) that uses dynamic programming to provide enhanced learning capabilities. First, the proposed approach examines multi-variable functions and delivers graph-based link predictions to locate the optimal nodes for edge networks. Moreover, it identifies a sub-path in multistage to continue data transfer if a route is unavailable due to certain communication circumstances. Second, while applying security, edge computing provides offloading services that lower the amount of processing power needed for low-constraint nodes. Finally, the SDS-GIoT model is verified with various experiments, and the performance results demonstrate its significance for a sustainable environment against existing solutions. IoT is a network of “things” that share and collect data from the environment. These “things” could be sensors, portable electronics, wearable technology, or any networked item that can carry out certain tasks ,2,3. SimThe proposed SDS-GIoT model offers the following contributions.It provides a data-driven approach for sustainable smart cities using multistage graph-based structures and improves the system’s response time.Intelligent decisions are made based on dynamic programming, which allows for effective computing with minimum complexity on the IoT networks.Edge computing and deterministic technique are combined to create and maintain system security. Using an offloading method lessens the burden of security measures on the devices.The proposed SDS-GIoT model is verified in terms of green computing metrics against existing work.This research paper is structured in sections. The development of information and communication technologies over the past few decades has sparked a trend toward smartening everyday things to improve human comfort. The paradigm of smart cities is a reaction to the objective of building future cities for quality assurance and sustainable development ,23,24. SThe authors in  developeA novel routing protocol termed Secured QoS aware Energy Efficient Routing Protocol is presented to improve WSN security and energy efficiency . Trust mThe technology of IoT networks with SDN architecture is being explored for the formation of smart cities. It senses the data from the targeted area and sends it to remote sites for processing and analysis. Many solutions have been recommended by researchers based on the aforementioned discussed work for coping with resource constraints with computing capabilities; however, the management of network data over the presence of huge traffic flow is one of the main research problems. Moreover, providing prompt responses on remote sites with affordable delay is also considered a significant research problem. It was also noticed that optimizing the routing process coped with the support of intelligent strategies; however, it exposed additional costs to the IoT devices as the network grew. This research proposed a reliable model for IoT technologies with the support of dynamic programming, leading to optimized efficiency. The states of the SDS-GIoT model are evaluated each time before announcing the optimal decision for data sending. Furthermore, edge computing integrates with blockchain technology to ensure data privacy and authentication. Edge computing provides an offloading method for decreasing the network burden in terms of applying security on the constraint devices.In this Section, we present the system model and detail of the SDS-GIoT model. The SDS-GIoT model comprises sensors, edge nodes, IoT devices, and wireless systems. IoT devices and sensors are utilized for collecting and transmitting environmental data. IoT devices and sensors explore the proposed routing algorithm for sustainable communication with minimum data damage. Additionally, the multi-variable objective function helps link predictions and facilitates dynamic programming for optimal decisions. The data is routed from the consistent and reliable nodes toward edge devices based on the decisions. The decisions are dynamic because they change each time the source node needs to send Route Request (RREQ). Later, the security layer is used to identify the potential threats in the IoT system. The main sub-sections of the SDS-GIoT model are discussed as follows.n. The nodes are arranged in the form of directed and weighted multistage Graph The proposed network model consists of sensors interacting with each other using wireless communication standards. The sensors denote by The sensor nodes can only communicate with edge devices and are not mobile.There are no resource restrictions on the network edges or sink nodes.At the edge of a sensor’s vicinity, edge devices are randomly positioned.No more nodes or devices can be included after deployment.Malicious nodes can generate false information and compromise the sending data and wireless channels.This Section provides a brief introduction to the states in the SDS-GIoT model. Multistage graph: This stage organizes the nodes in the form of multiple stages, and stages are interconnected with edges.Tables’ initialization: In this state, nodes’ information and network conditions are recorded along with the identities of devices.Iterative function: The repeated function is performed to determine the optimal routing strategy in this state.Edge cost: Nodes compute the cost, and accordingly, the minimum value offers the optimal decisions. In case the outcome is not optimal, then the iterative function is executed again.Authentic nodes: This state determines the validity of devices in terms of authentication. If nodes are declared authentic, then communication is allowed by the system; otherwise, alert messages are recorded in the local tables.Secured sessions: In this state, the system attains data privacy with integrity using session keys and security methods.In this Section, we present a novel solution for routing the IoT data with the support of dynamic programming. The sensors sense the surrounding data and execute the next-hop selection process in a distributed way, unlike most of the proposed solutions that do not track the congestion and traffic-related information in the decision system. Our proposed data-driven model keeps such information while reaching an optimal decision. Accordingly, the SDS-GIoT model offers a sustainable approach to attaining a timely wireless system with load balancing. In the SDS-GIoT model, the nodes are arranged in the multistage graph such that each node has some neighbors until it reaches the destination state. We consider states are denoted by In Equation (1), To evaluate priority A connection is not taken into account by the decision-making algorithm if the According to Equation (4), when the queued packets increase, the priority value of Afterward, the SDS-GIoT model develops a security algorithm for devices with limited constraints. In this phase, edge devices act as a central hub for their associate sensors, and they help authenticate the peer devices before data transmission. Let us consider that Upon decrypting the message, Later, using received keys, the SDS-GIoT model performs data encryption on data blocks Algorithm 1: Data-driven secured optimization model using dynamic programmingProcedure Sec_data_drivenStep 1: Network-setupStep 2: Multistage graphs with nodes and edgesStep 3: Compute the traffic by exploring Step 4: Compute Step 5: Cost using objective function Step 6: Threshold evaluation for sending route request Step 7: If the neighbor state is not equal to the destination thenRepeat Steps 3 to 6End ifStep 8: Performs network authenticationStep 9: If authenticity is verified then data transmissionElseDrop the request packetEnd ifStep 10: Generate random keys and perform a security functionStep 11: If all data packets are delivered to the destination thenSend ACK to the source deviceElsePerform incremental encryptionEnd ifStep 12: End procedureStep 1: This process continued in the form of various increments for maintaining the integrity and privacy of data blocks. This Section describes the network scenarios and the performance results of the SDS-GIoT model with MADCR and SRPMA. We perform various tests in two scenarios. One is varying IoT devices and the second is varying data generation rates. IoT devices range in number from 25 to 125, and data generation rates range from 1000–5000 bits/s. The varying number of nodes is considered in experiments to identify the effectiveness of the SDS-GIoT model under different sizes. The data generation rate indicates the amount of data that is produced by the nodes with time. The size of the control packet is set to 256 bits. We consider 10 edge devices and 2 sink nodes. The initial energy of nodes varies from 3 j to 6 j. The distance between nodes is measured using Euclidean distance. All the devices have limited constraints except edge devices and sink nodes. The simulations are carried out using NS-2.35 on the Ubuntu platform. The network dimension is set to 1000 m × 1000 m. A total of 30 simulations were run to take an average of the performance metrics. The transmission power of the IoT nodes is set to 10 m. Furthermore, varying malicious nodes are deployed in the simulation environment. The data were obtained from trace files after various simulations, and stored in log files for further analysis and determining the efficacy of the SDS-GIoT model. This Section provides the simulated graphs and their discussion of the SDS-GIoT model and other related studies. The performance is evaluated for network throughput, latency, packet drop ratio, and the maximum number of rounds in a green IoT system. The proposed SDS-GIoT model remarkably improved green computing using the dynamic structure and the least overhead. It consumes less energy in sensing, aggregation, and transmission phases with the support of intelligent decision systems. The usage of multistage graphs explicitly shows a better outcome for the proposed sustainable solution in the light of dynamic programming. Moreover, the authentication and security operation also protect the green system by exploring lightweight cryptographic operations. The SDS-GIoT model maintains the efficiency of IoT resources in terms of energy constraints and increases its performance as compared to existing work. Unlike the majority of the existing work, which does not consider the intelligent approaches for the identification of suitable nodes and links in the data forwarding processes, to determine the most reliable circumstances and make the IoT system more sustainable, the proposed SDS-GIoT model makes use of dynamic programming. In The performance of the SDS-GIoT model is evaluated in comparison with existing approaches. The performance of the SDS-GIoT model in comparison to the existing approaches is shown in A comparison of the SDS-GIoT model with alternative studies is shown in Recently, the development of a sustainable IoT system solution using a combination of storage and communication devices has experienced rapid growth. Although IoT nodes are self-configuring and inexpensive, they have energy, transmission, and memory limitations. Most of the solutions have been vulnerable to numerous network threats and compromised information as a result of faulty communication connectivity. This paper presents a communication protocol for an IoT-based system using dynamic programming in multistage graphs and extracts the optimal information for the transmission of network data. Additionally, it decreases the response time and efficiently manages the nodes’ energy resources. Additionally, the proposed security mechanism is controlled by edge devices without additional overhead to the IoT nodes. The secret keys and data blocks are securely forwarded toward the sink using authentic blockchain technologies. The simulation results have proven the efficacy of the proposed protocol with related studies in terms of performance metrics. However, it was observed that moving the IoT nodes from their initial deployment points causes frequent data interruption and connectivity issues. As a result, the loss rate of packet reception increases. In the future, we aim to improve the proposed protocol in terms of the mobility model and would like to integrate deep learning intelligent techniques for coping with distributed network attacks.


Using this covariation, three groups with distinct response patterns to bumetanide were detected, including the best , the least  and the medium  responding groups. Including the cytokine levels significantly improved the prediction of the best responding group before treatment  compared with the model without the cytokine levels . Cytokine measurements can help in identifying possible responders to bumetanide in ASD children, suggesting that immune responses may interact with the mechanism of action of bumetanide to enhance the GABA function in ASD.Bumetanide, a drug being studied in autism spectrum disorder (ASD) may act to restore gamma-aminobutyric acid (GABA) function, which may be modulated by the immune system. However, the interaction between bumetanide and the immune system remains unclear. Seventy-nine children with ASD were analysed from a longitudinal sample for a 3-month treatment of bumetanide. The covariation between symptom improvements and cytokine changes was calculated and validated by sparse canonical correlation analysis. Response patterns to bumetanide were revealed by clustering analysis. Five classifiers were used to test whether including the baseline information of cytokines could improve the prediction of the response patterns using an independent test sample. An immuno-behavioural covariation was identified between symptom improvements in the Childhood Autism Rating Scale (CARS) and the cytokine changes among interferon (IFN)- Autism spectrum disorder (ASD) affects about 1% children around the world . Currentγ can decrease the levels of NKCC1 and the α-subunit of Na+-K+-ATPase, contributing to the restore of inhibitory GABA function . To date, the detailed results of these Phase 3 trials have not been published, we could not learn more details about the operation and the outcomes. Considering the nature of heterogeneity in the aetiology and pathology of ASD, the development of biological first-line treatment may only be effective in certain subtypes [It was disappointing that the phase 3 clinical studies assessing bumetanide in the treatment of ASD were terminated after the middle analysis, which showed no sign of effectiveness, led by two pharmaceutical companies Servier and Neurochlore [subtypes . Therefosubtypes . Multiplsubtypes , 19, 48.subtypes . HoweverThere are several limitations of our study. Although we had two separate data sets to validate our findings, the sample sizes were limited. Previously a sex difference in the cytokine-symptom association had been reported, but we could not test such sex difference owing to the small numbers of girls with ASD in our sample . Hence, In summary, we identified an association between the changes of the cytokine levels and the improvements in symptoms after the bumetanide treatment in young children with ASD, and found that the treatment effect of bumetanide can be better characterised by an immuno-behavioural covariation. This finding may provide new clinically important evidence supporting the hypothesis that immune responses may interact with the mechanism of bumetanide to restore the GABAergic function in ASD. This finding may also have relevance for determining enriched samples of ASD children to participate in novel drug treatment studies of drugs with a similar mode of action to bumetanide, but with potentially greater efficacy and fewer side effects.Supplementary Materials


Healthcare challenges in low- and middle-income (LMICs) have been the focus of many digital initiatives that have aimed to ensure consistent implementation of these services. During the COVID-19 pandemic, several lockdowns were imposed globally by government authorities to contain the spread of the virus. This triggered a rapid effort to integrate digital technologies into the existing health systems of LMICs . This research used the Activity Based Costing—Ingredients (ABC-I) method approach with aims to break down the program into a sum of mutually exclusive and exhaustive activities. A brief review by Yogesh and Karthikeyan, discussed the future trends and directions in health informatics, noting that there are no proven design blueprints for a comprehensive infrastructure. The authors also report that big data is playing an important role in health informatics, where a large amount of data related to healthcare is generated.One of the challenges of implementation of digital health in LMICs is cost. A detailed protocol on determining the costs of a large mHealth job aid for health and nutrition in India is discussed by Senjam and Primo, focused upon the challenges and enablers for smartphone use by persons with vision loss during the COVID-19 pandemic. The most important enabling factors found were the presence of a screen reader, data connection of the mobile and the ability to assess multiple languages. Conversely, frequent challenges included poor battery backup, frequent unwanted ads or pop-ups unreadable by a screen reader, and slow or unresponsive screen readers. In a study from Nepal, Sankhi et al.  that was used for the tele-examination for patients with disabilities at a remote care center. The same patients were examined by two independent sonographers using 5G-based robot-assisted remote ultrasound. The authors concluded that the use of a 5G-based robot-assisted remote ultrasound system is feasible in patients with disabilities at a remote care center and results in similar diagnostic efficacy to traditional bedside ultrasound, the gold standard. A similar article by Lim et al., evaluated the reliability and accuracy of 2D photogrammetry, as compared to direct measurement (gold standard). The authors discuss that three facial dimensions cannot be measured reliably and accurately using the 2D photogrammetry method because of poor inter-rater reliability of 2D photogrammetry.Another research article by Jain et al., reported the success of monitoring and holistic care of healthcare workers affected with mild COVID-19 and residing under home isolation through the use of digital technology. Healthcare workers faced additional challenges when compared to the general population besides a greater risk of becoming infected, harboring a higher virological burden, and a potentially more serious illness, they have additional work-related and psychosocial stressors.In another study on remote monitoring Taken together, this collection of articles highlight the need to test the validity and reliability of digital health tools to streamline their function and design them according to the needs of programs in low income countries in order to minimize implementation challenges. Policymakers must consider usefulness, usability, integration, and infrastructure issues to improve their digital health functions. For full-scale sustainability, financing for all aspects of digital health solutions needs to be integrated into routine health budgets and the budgeting processes.MD wrote the editorial. All authors co-edited the Research Topic, contributed to the editorial editing, and approved the submitted version.


Recent advances in assisted reproductive technology (ART) have increased the effectiveness of fertility treatments. Some examples of ART include the storage of gametes and embryos, in vitro fertilization (IVF), artificial insemination (AI), and embryo transfer (ET). The storage of gametes, especially sperm, is a popular method facilitating the reproductive success of farm, companion, working, and wild-living animals. The choice of the preferred sperm-preservation method (liquid or cryopreservation) is determined by the species. Therefore, species-specific characteristics and the influence of various factors, such as storage temperature and time, or the type of diluent, have to be considered during the improvement of sperm-preservation methods. Cryopreservation of ejaculated and epididymal sperm supports the long-term preservation of valuable genetic material for reproductive purposes. The quality of preserved sperm is assessed with the use of various laboratory techniques . The optimal preservation technique should maintain the fertilizing ability of spermatozoa, which is evaluated based on the results of laboratory analyses and confirmed by the success rate of in vitro and AI procedures.Assisted reproduction technologies are applied mainly in mammals and less commonly used in birds and other taxonomic groups. Semen quality differs between species and individuals of the same species. The sperm of various animal species differ in morphology, biological parameters, and susceptibility to cryopreservation . InterspNumerous factors influence the quality and fertilizing ability of liquid-preserved and cryopreserved sperm. The quality of fresh semen is one such factor. Morphological defects can undermine sperm cells’ suitability for liquid preservation and cryopreservation. Henning et al.  reportedProlonged sperm storage in a liquid state can induce considerable changes in the morphology and function of spermatozoa. Numerous morphometric changes in the head and tail were observed in boar spermatozoa preserved in a liquid state at a temperature of 17 °C . These cSemen’s suitability for cryopreservation is also determined by the animal’s age. Ejaculate volume and quality change with age and can affect sperm cells’ resistance to freezing. The quality of cryopreserved bull sperm was influenced by differences in the proteomic composition of semen . The semThe choice of a suitable extender and the optimal temperature during liquid storage can enhance sperm viability and functionality. During storage, the temperature is decreased to 4–5 °C to increase the viability of sperm cells from various animal species ,7. Low tCervid spermatozoa are preserved with the use of extenders designed for other ruminants (cattle and sheep). Differences in extender composition can significantly affect the viability and functionality of European red deer epididymal spermatozoa stored at a temperature of 5 °C . An egg Cryopreservation is the most effective technique for the long-term conservation of valuable animal genetic resources ,9,10. HoIn addition, cryopreservation contributes to oxidative stress which leads to the production of reactive oxygen species (ROS) that can damage various structures in sperm cells . The addCryopreservation is most often applied in mammals and birds used as livestock . This prCastillo et al.  proposedPituophis ruthveni) [Little is known about the effectiveness of sperm cryopreservation in other taxonomic groups, such as reptiles. Sperm conservation was moderately successful in several snake species. However, research has shown that selected cryoprotective agents and post-thaw treatments can enhance the quality of cryopreserved sperm from the endangered Louisiana pine snake (uthveni) . The resNumerous factors, including species-specific differences, quality of fresh semen, conservation methods , and the animal’s age, have to be considered in the process of developing effective semen preservation techniques. Each of these factors affects the quality of preserved sperm. The articles published in this Special Issue have significantly expanded current knowledge on liquid preservation and cryopreservation of sperm from various animal species . The presented studies have also contributed new information about the quality and preservation of sperm from wild-living animals.


In the past decade, we have witnessed the introduction and dissemination of transoral robotic surgery for the treatment of tumors, mainly of the oropharynx and larynx. The use of robotic surgery improves visualization of the operative field due to its three-dimensional image and enhances the surgeon's dexterity due to bimanual control of the robotic arms. Furthermore, the assistant contributes with suction and tissue traction, which leads to the use of four instruments during surgery, something impossible during a transoral resection through laryngoscopy, for instance.Therefore, this study reports the first case of supraglottic partial laryngectomy performed by transoral robotic surgery in Brazil, as well as documents the late oncologic and functional results (Approved by the Research Ethics Committee under No. 228/14).A 57-year-old female patient was evaluated for a four month complaint of odynophagia; she was a long-term smoker (30 pack-years) and a non-alcoholic. Physical examination revealed no lesions at the oroscopy and no palpable cervical lymph nodes. The nasofibrolaryngoscopy identified a large vegetating lesion affecting the entire epiglottis and extending to the left aryepiglottic fold, but not affecting the arytenoid fold or the left ventricular fold; both vocal folds were still mobile.An incisional biopsy revealed that the lesion was a moderately differentiated squamous cell carcinoma (SCC). Assessment by computed tomography  showed tdaVinci SI Surgical System®  equipment  was performed, and it retrieved 11 articles,In this case, some aspects are noteworthy and were later verified by other studies summarized here: the patient had an uneventful postoperative period, in addition to very satisfactory oncologic and functional results. The desire to provide the patient's late follow-up status led to the delay in reporting the present case.This case describes the viability of supraglottic partial laryngectomy by transoral robotic approach, with good postoperative evolution and early rehabilitation. It is therefore a safe method, with very satisfactory oncologic and functional results.The authors declare no conflicts of interest.


To explore the challenges faced by medical colleges and coping strategies used in implementing accreditation standards by Pakistan Medical Commission during accreditation inspection 2019.In this qualitative case study, four medical colleges and their affiliated hospitals from three cities in Khyber Pakhtunkhwa province of Pakistan were selected through purposive sampling. Data was collected through focus group discussions (FGD) through Open-ended questions, based on CIPP  model. Each focus group comprised of Dean, the Director Department of Medical Education (DME) and the Medical Director of the hospital. Data were thematically analyzed and results were based on the CIPP model.administrative challenges, accreditation challenges and resource challenges. The administrative challenges theme was further explained under subthemes of rules and regulation challenges, documentation and record challenges, and DME-related challenges. The accreditation-related challenges theme was explored in-depth with subthemes of accreditation process-related challenges, accreditation standards-related challenges and curriculum-related challenges. The resource challenges theme was described under sub-themes of infrastructure-related challenges, human resources and financial challenges. The commonest coping strategies adopted by medical colleges were establishing DME, emergency preparatory meetings of staff, and hiring staff on an emergency basis, to overcome administrative, accreditation and resource challenges respectively. Future suggestions for improving the accreditation process in the local context were highlighted.Three themes identified were Main challenges identified were administrative challenges, accreditation challenges and resource challenges. Coping strategies by the medical colleges for these challenges are highlighted. The accreditation body should harmonize the process of accreditation with medical colleges and other stakeholders. The surge in global demand for health professionals and the resulting exponential increase in the number of health professions education institutions has blurred the international boundaries for medical students and physicians. The wide variations in medical training programs and the lack of robust accreditation systems have threatened the overall quality of medical education worldwide. Globally, there is increasing realization for developing global medical education standards and accreditation systems to ensure the quality of medical education. World Federation for Medical Education (WFME) published its global standards to guide countries to contextualize and develop their own standards.2Accreditation is one of the contemporary methods for quality assurance in many fields including medical schools.7Pakistan Medical Commission (PMC), as the sole regulatory body carries the responsibility of accreditation for over 150 medical and dental colleges in the country. The country requires a robust accreditation system to cater the needs of such a large number of medical and dental colleges. Few important features of the system include developing and updating accreditation standards that are contextual to the needs of the country, developing policies and procedures for accreditation in consultation with key stakeholders, and ensuring that faculty and other staff are well-trained to prepare the colleges for accreditation. Research on medical accreditation is limited in Pakistan. WFME standards provide a good start to develop a robust accreditation system in the country. In 2019, Wajid et al. conducted a study to assess the relevance of WFME standards to the Pakistani context.9No study has been conducted in Pakistan on the experiences of key stakeholders  about the accreditation challenges faced during the accreditation process. This study provides evidence from the first-hand experiences of key persons involved in the accreditation process, challenges faced by them while preparing the institution for accreditation and strategies adopted by them for overcoming these challenges.This qualitative case study was conducted in 2020-2021, after the PMC inspection in 2019 for accreditation of medical colleges. The study was approved by the ethical committee of Khyber Medical University Peshawar, Pakistan [Reference No. DIR/KMU.EB/CF/00077 ON 06/09/2021].. Administrative approvals from the concerned medical colleges and written, informed consent from all participants were taken.Four medical colleges and their affiliated hospitals from three cities in the Khyber Pakhtunkhwa (KP) province of Pakistan were selected through a purposive, maximum variation sampling technique. The medical colleges were selected from the central, northern and southern districts of KP. Out of four medical colleges, three were public sector and one was a private medical collegeContext, Input, Process & Product) model was used as a theoretical framework because of its inherent capacity for objective conclusions.context and input evaluation describe the challenges faced during the preparatory phase of inspection. The process evaluation describes challenges faced during the actual inspection and product evaluation is about various coping strategies and future suggestions. Completion of the accreditation process was taken as the end product. A questionnaire, based on the CIPP model was developed comprising of eight open-ended questions, covering all the components of CIPP.The CIPP (Data was collected through focus group discussions (FGDs). Two FGDs were conducted face to face in medical colleges and another two, through Zoom meetings because of the Covid-19 pandemic. Each FGD lasted for 45-60 minutes. Each focus group comprised of Dean, DME director of each medical college and medical director (MD) or medical superintendent (MS) of their affiliated hospital. Proceedings of FGD were recorded and notes were taken for important points.Audio recording of FGDs was transcribed manually and was counter-checked by an independent observer. The data was analyzed through three cycles of coding and thematic analysis was done to generate themes from these codes. The results were analyzed and compiled based on the CIPP model.Among the 12 FGD participants, 3 (25%) were females and 9 (75%) males aged from 40-50 years. The main challenges identified were grouped into three themes & nine subthemes. Theme one included administrative challenges with subthemes of rules and regulation challenges, documentation and record challenges and DME-related challenges .The second theme was accreditation challenges with sub-themes of accreditation process-related challenges, accreditation standards-related challenges and curriculum-related challenges . The thiChallenges faced by medical colleges during PMC accreditation inspection 2019 were grouped under three themes: administrative, accreditation and resource challenges.administrative challenges at the level of PMC, medical colleges, and hospital administration during the accreditation process. These challenges mainly stem from the lack of planning, lack of coordination among different stakeholders and poor staff training for preparing and conducting accreditation. The PMC, since its inception struggled in developing and implementing its rules, regulation and policies.14The study identified several The medical colleges and hospital administration faced various executive challenges due to variations in the administrative and financial rules and regulations of the institutions working under the Medical Teaching Institution (MTI) act and the non-MTI act. In 2015, KP Government granted financial and administrative autonomy to various medical colleges and affiliated teaching hospitals through the MTI act to improve the quality of health services and delegated administrative powers and responsibilities to an independent policy board.Though the department of medical education (DME) was declared the accreditation of institutions in 2008, the accrediting body did not provide the regulations and policy guidelines for establishing DMEs.One of the biggest challenges faced by almost every medical college was a poor documentation system. Short preparatory time, variation in rules and regulations and non-familiarity with new standards resulted in an increased workload on faculty and hospital administration. Similar accreditation challenges of increased workload, faculty resistance and too many standards were reported in a study from Iran.19Participants of our study mentioned several coping strategies to get through the accreditation process successfully. Most strategies were mere box-ticking exercises to maximize accreditation scores, rather than addressing the gaps in the quality of education in a more transformative and sustainable manner. Few strategies included establishing DME , manipulation of records and creating new documents on an emergency basis.accreditation body and the planning and process of accreditation. Proficient and skillful inspectors are vital to conducting the accreditation survey effectively. Inspectors must be trained well on the nature and measurement of standards, and their role during the accreditation process. Unfortunately, the PMC overlooked both the selection and training of its inspectors, which resulted in an inconsistent inspection process. One participant reported that the inspectors finished the whole inspection process in a college in single day only . Such threat to the validity of the accreditation process has been reported in other studies from Pakistan and Iran.Some of the challenges faced during the inspection were solely related to the Coping strategies included calling emergency staff meetings to distribute responsibilities to prepare the college for accreditation, staff orientation on accreditation standards and identifying the major deficiencies that could drastically pull the score down. It was evident that there was no concerted quality improvement effort behind the accreditation process. The curricular issues were addressed by medical colleges through the capacity building of faculty members. Developing new teaching strategies, incorporation of study guides, adjustment of subject hours in timetable, improved internal assessment and relevant documentation were the main reforms in the curriculum.resource challenges: human resources, infrastructure, and financial difficulties. PMC has set standards for human resources in medical colleges and affiliated hospitals. These resources include a range of faculty, office personnel and other support staff. Medical colleges generally faced the challenges of fulfilling human resource requirements as per PMC standards. Studies conducted in PakistanMedical colleges and hospital administration faced various Participants gave several suggestions to improve the accreditation process. These related to the composition of the accreditation team, inspection schedule, inspectors training, liaison between health and finance departments, better coordination between the colleges and PMC and post-inspection analysis. The inspection of an institution for accreditation purposes is a resource intensive process. PMC may consider introducing a pre-inspection process to guide the institutions conduct their self-assessment for readiness for inspection. Effective staff training at all levels is the key to successful accreditation.This study was conducted in just four medical colleges of KP province, so it cannot be generalized to all medical colleges of Pakistan because of different administrative and financial rules and regulations in other provinces. The accreditation challenges could be explored more holistically by interviewing the heads of the inspection committees and conveners of the accreditation team. Medical colleges in the public and private sectors may also need to be explored further as the challenges may be different for the two sectors.The main challenges faced by medical colleges during accreditation inspection were related to administration, accreditation body and resources. The main administrative challenges faced were related to records and documentation followed by highly variable regulations among public and private sector medical colleges and hospitals. The significant resource-related challenges were meeting human resource requirements, poor infrastructure and financial constraints. Untrained inspectors, poor inspection scheduling and lack of uniform inspection standards were the accrediting body related challenges.The study provides evidence not only about the challenges during the accreditation process but also shows how institutions view accreditation as a policing and audit process. Quality assurance is the essence of any accreditation process. PMC needs to develop a robust accreditation system in consultation with the stakeholders. An accreditation department at PMC that is equipped with technically qualified staff and leadership support to develop and implement an accreditation system in over 150 medical and dental colleges of the country is perhaps the biggest challenge faced by the Commission and the colleges. Effective stakeholder coordination and staff training at all levels may significantly improve the accreditation system in Pakistan.FG: Conceptualization, data collection, data analysis. Writing the manuscript and final editing of the manuscript. Approval of the final manuscriptUM: Conceptualization, developed methodology, analyzed data, critical revision manuscripts and final editing of the manuscript. Approval of the final manuscriptGW: Conceptualization, developed methodology, critical revision of all drafts and final editing of the manuscript. Approval of the final manuscriptAS: Data collection and analysis. Manuscript writing. Approval of the final manuscriptAll authors agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.


This paper presents a new framework for lightweight optimisation of functionally graded lattice structures (FGLSs) with a particular focus on enhancing and guaranteeing structural safety through three main contributions. Firstly, a design strategy of adding fillets to the joints of body-centred cubic (BCC) type lattice cells was proposed to improve the effective yield stress of the lattices. Secondly, effective properties of lattice metamaterials were experimentally characterised by conducting quasi-static uniaxial compression tests on selective laser melted specimens of both Ti6Al4V BCC and filleted BCC (BCC-F) lattices with different relative densities. Thirdly, a yield stress constraint for optimising FGLSs was developed based on surrogate models quantifying the relationships between the relative density and the effective properties of BCC and BCC-F lattices developed using experimental results assisted by numerical homogenisation. This framework was tested with two case studies. Results showed that structural safety with respect to avoiding yield failure of the optimised FGLSs can be ensured and the introduction of fillets can effectively improve the strength-to-weight ratio of the optimised FGLSs composed of BCC type lattices. The BCC-F FGLS achieved 14.5% improvement in weight reduction compared with BCC FGLS for the Messerschmitt-Bölkow-Blohm beam optimisation case study. Metamaterials are materials engineered to exhibit designed properties. Lattice metamaterials are biomimetic lightweight materials that are usually constructed by repeating networks of lattice cells . The desExperimental investigations of deformation behaviour and effective mechanical properties  of lattice metamaterials have been conducted in a number of studies. The investigations are usually carried out by conducting quasi-static uniaxial compression tests on lattice metamaterials with various cell topologies and different parent materials  at a range of relative densities. For instance, Crupi et al.  investigMeanwhile, lattice metamaterials have also drawn great interests from researchers on computational mechanics to develop recent advances from the structural design and optimisation perspective. This is mainly motivated by the potential of functionally graded lattice structures (FGLSs) in achieving superior functional performances, by efficiently tailoring the distributions of lattice configurations. Owing to the regular arrangement of struts in a lattice cell, the geometry of a lattice cell can be described by a limited number of geometric parameters, such as strut diameters. Thus, the effective material properties  of lattice metamaterials can be engineered by controlling the geometric parameters. Optimisation frameworks have been developed using such geometric parameters as design variables to obtain the optimal design of FGLSs. For example, Takezawa et al.  adopted To bring the research on the design of FGLSs closer to applications, imposing failure constraints, such as yield stress constraints, is crucial, in terms of ensuring the structural safety. The von Mises yield criterion is widely adopted in formulating the yield stress constraint in structural optimisation, such as topology optimisation ,25,26, aBy replacing the numerical homogenisation with the direct experimental characterisation of lattice metamaterials, the effects of manufacturing on the effective properties of lattice metamaterials can be considered, which obviously means the performance of lattice structures can be more accurately predicted in simulations, thus the optimised structure can be more reliable. However, the fact is that few of the existing studies on the optimisation of FGLSs have adopted an experimental approach to determine the effective properties of lattice metamaterials, just like research focused on experimental studies of AMed lattices has rarely applied their findings to the structural design and optimisation of the lattice structures. Therefore, to facilitate the development of FGLSs towards real applications as high value-added components, the synergy between experimental and numerical studies and a holistic methodology for designing such a new family of structures are highly desired.In this paper, we present a new optimisation framework for numerical optimal design of high-stiffness and lightweight FGLSs, with a unique focus on improving and ensuring structural safety by innovatively introducing joint fillets to the lattice cell design for increased effective yield stress, integrating the experimental testing of lattice metamaterials as a new step for accurately characterising effective properties, and adding a yield constraint to the optimisation to avoid yield failure of the optimised FGLSs. A typical bending dominated lattice type, body-centred cubic (BCC), as well as the representative parent material and AM approach, Ti6Al4V and selective laser melting (SLM), respectively, are adopted in this study to demonstrate the framework and methodologies. The abbreviations used in this paper are tabulated in Due to the unconventional nature of the study and its workflow, the structure and methodology of this work is firstly overviewed in this section. Subsequently, the geometries of BCC type lattice unit cells for the lattice metamaterial were designed in this section. Fillets were introduced to the joints of lattice cells to reduce stress concentration. The effects of fillet radii on the effective yields stresses of the BCC type lattices were investigated through a parametric study using numerical homogenisation. The procedures for experimental characterisation of the effective material properties  of the BCC and BCC-F lattices are presented in this section.This subsection overviews the structure and methodologies adopted in this paper. The flowchart in s (PBCs)  applied The BCC lattice cell type is a bending dominated lattice type ,36,37 anAdding fillets to lattice joints results in adding extra material to lattice cells. The admissible space in a lattice cell for adding fillets is restricted by the topology and the strut dimeter of the lattice cell. In general, a lattice cell with a higher strut diameter leaves a smaller space for adding fillets to its joint. Thus, in this study, the assumption was made that the strut diameter, In this study, the reciprocal relationship between The effect of  EasyPBC  in AbaquTo determine the optimal value of It has been discussed in Different from the numerical homogenisation method with PBC applied to a single lattice unit cell, the direct experimental characterisation was conducted on actual lattice structures, architected by a sufficient amount of repeatedly arranged identical lattice cells for each lattice design configuration. Each lattice metamaterial specimen was designed to be a cubic containing 512 (8 × 8 × 8) lattice cells with a uniform cell dimension of 5 × 5 × 5 mm, resulting in the total dimension of a lattice metamaterial specimen being 40 × 40 × 40 mm. The CAD models of specimen designs of the BCC and BCC-F lattices with different designed The specimens were fabricated by using SLM with the same parent material, Ti6Al4V, as given in The reason for designing the maximum relative density of the lattice metamaterials to be 0.51 in this study is briefly explained as follows: it was observed that specimens with a higher relative density fractured after fabrication. The two fabricated groups of specimens with different measured relative densities, Uniaxial compression tests were conducted to characterise the effective mechanical properties of the fabricated BCC and BCC-F lattices. The effective mechanical properties to be experimentally measured include the effective Young’s modulus, the effective Poisson’s ratio, and the effective yield stress.The experimental setup is shown in The experimental results of the uniaxial compression tests on BCC and BCC-F lattices are presented and discussed in this section. Based on the experimental results, the effective material properties  of BCC and BCC-F lattices were characterised and evaluated. Subsequently, surrogate models of both lattices were developed by adopting the experimental characterisation results with the assistance of numerical homogenisation simulations to quantify the relationship between relative density and effective material properties of the lattices. The surrogate models were then used to develop a yield stress constrained optimisation framework for the design of functionally graded lattice structures. The framework was tested with an L-shaped beam bending case study and a Messerschmitt-Bölkow-Blohm (MBB) beam bending case study.All the experimental results are presented based on the measured relative density values in this section, hence, for the purpose of brevity the relative density refers to the experimentally measured relative density in the following contents of this section if not annotated. The experimental results of the engineering stress–strain curves of the BCC and the BCC-F specimens at different relative density levels are shown in study of . When thFor the BCC-F specimens (composed of filleted lattices), when the relative density ttice in , where ty et al. , with thBecause the aim of the uniaxial compression tests was to characterise the effective material properties of the lattice metamaterials for the purpose of developing surrogate models used in the lattice structural optimisation, a detailed discussion on the fracture modes of lattice metamaterials is out of the scope of this research. This study focuses on the elastic regions and the yield stresses of the stress–strain curves. Good repeatability of the stress–strain curves before yield was observed from repeated tests (on three duplicated specimens). The effective Young’s moduli, effective Poisson’s ratios, and effective yield stresses of the BCC and BCC-F lattices with different relative densities will be evaluated and compared in the next subsection.The effective Young’s moduli were obtained by measuring the slopes of the stress–strain curves in the elastic deformation domains. The effective yield stresses were obtained by measuring the 0.2% offset stresses of the stress–strain curves. The effective Poisson’s ratios were obtained by measuring the vertical compressive strains and the horizontal expansion strains in the elastic deformation domains, where the strains were measured by adopting the digital image correlation method.Both the experimental and numerical homogenisation results of the relative effective Young’s modulus, the relative effective yield stress, and the effective Poisson’s ratio of the BCC and the BCC-F lattices are shown in For the experimental results  was solved using the finite element (FE) method. Similar to many FE-based structural optimisation frameworks, a checkerboard-like pattern of alternating material and void can be easily generated in optimised structures . This chThe upper bound, U is a function of The effective elasticity tensor with the design variable filter and the threshold function applied was adopted to calculate the stress tensor in the yield stress constraint. The stress tensor can be written as:pn is the exponent of the p-norm of pn, the inequality constraint can be written as:The maximum value of Hence, the optimisation problem Problem An optimisation platform was developed to implement the optimisation of FGLSs. A flowchart of this platform is demonstrated in Two case studies are presented in this section to test the capability of the proposed yield constrained lattice structural optimisation framework. The first case study was an L-shaped beam bending case, in which the optimisation problem As mentioned in the previous section, the FE analysis and sensitivity analysis of optimisation problems were carried out in Nastran 2018. The sensitivity analysis was conducted using the modified method of feasible directions (MMFD) ,48. MMFDThe dimensions and boundary conditions of the L-shaped beam case study are demonstrated in The optimisation results of the distributions of relative density and von Mises stress of the BCC-F L-shaped beam are shown in The volume proportion histogram of stress constraint measurement  has been developed in this paper, with a particular focus on enhancing and guaranteeing structural safety through three contributions: firstly, from the lattice cell design perspective, new fillet features have been added to the joints of BCC lattice cells to reduce stress concentration; secondly, from the lattice metamaterials characterisation perspective, the effective material properties (effective elasticity tensors and effective yield stresses) of BCC and filleted BCC (BCC-F) lattices have been experimentally investigated and characterised to mitigate the discrepancies between the numerically characterised and the actual effective material properties; thirdly, a von Mises yield stress constraint for the optimisation of FGLSs has been developed based on the surrogate models of the effective material properties of the BCC and BCC-F lattices, developed using the experimental results with the assistance of numerical homogenisation simulations. The proposed framework has been implemented by establishing an optimisation platform based on MATLAB and Nastran and tested through an L-shaped beam bending case study and an MBB beam bending case study.The key findings of this paper are summarised as follows: (i) The effective yield stress of the BCC type lattice metamaterials increases monotonically with the increase of fillet parameter, To broaden the applications in the future, the framework developed in this paper can be extended to include a wide range of bending dominated lattice metamaterials, such as primitive cubic lattices.