Hydrogel composites can be prepared from cellulose-based materials and other gel materials, thus combining the advantages of both kinds of material. The aerogel, porous material formed after removing the water in the hydrogel, can maintain the network structure. Hydrogel and aerogel have high application potential. However, low mechanical strength and weight loss of cellulose hydrogel due to the water dehydration/absorption limit the feasibility of repeated use. In this study, cellulose hydrogels were prepared using microcrystalline cellulose (MC), carboxymethyl cellulose (CMC), and hydroxyethyl cellulose (HEC) as raw materials. Waterborne polyurethane (WPU) was added during the preparation process to form cellulose/WPU composite hydrogel and aerogel. The influence of the cellulose type and WPU addition ratio on the performance of hydrogel and aerogel were investigated. The results show that the introduction of WPU can help strengthen and stabilize the structure of cellulose hydrogel, reduce weight loss caused by water absorption and dehydration, and improve its reusability. The mixing of cellulose and WPU at a weight ratio of 90/10 is the best ratio to make the cellulose/WPU composite aerogel with the highest water swelling capacity and heat resistance. Hydrogel is a polymeric network material with low crosslink density, which has swelling ability and retains a large amount of water in the structure but is not soluble in water or solvents ,2,3. HydCellulose is highly hydrophilic, and the pores within the network structure can hold a large amount of water. The cellulose aerogel formed by dehydration of cellulose hydrogel has the potential to allow repeated water absorption and dehydration ,19. Our N,N’-methylene bisacrylamide as a crosslinking agent to obtain MC hydrogel. This cellulose hydrogel was immersed in a mixed aqueous solution of ferric trichloride and sodium p-toluenesulfonate, and finally immersed in the pyrrole solution to form a semi-interpenetrating cellulose/polypyrrole hybrid hydrogel. The results indicate that the hydrogel has conductivity and can improve the mechanical strength of cellulose hydrogel [N,N’-methylene-bis-acrylamide as a crosslinking agent and ammonium persulfate as an initiator to prepare CMC/AM hybrid hydrogel. The aerogel was obtained by freeze-drying of hydrogel. They produced a gel material with better mechanical properties and ability to adsorb heavy metals [Liang et al. (2015) dissolved microcrystalline cellulose (MC) in 1-butyl-3-methylimidazolium chloride ionic solution and used hydrogel . Godiya y metals . Wang ety metals . Tang ety metals . Verdoloy metals .PU is a resin with a urethane structure obtained by reacting the NCO group of an isocyanate with the OH group of a polyol. Due to its good toughness, ductility, strength, wear resistance, and weather resistance, PU resin is widely used in adhesives, coatings, printings, biomedical polymers, architectural engineering, the automobile industry, textile processing, and other fields ,28. HoweHowever, to date, few studies have combined cellulose hydrogels and aerogels with WPU resins ,33. WPU Microcrystalline cellulose , carboxymethyl cellulose , hydroxyethyl cellulose , sodium hydroxide , urea , epichlorohydrin , polytetramethylene ether glycol , isophorone diisocyanate , dimethylolpropionic acid , dimethylacetamide , ethylenediamine , and triethylamine .2O and NaOH/H2O with weight ratios of 4/6/90 and 4/96, respectively, were used as dissolving reagents. The cellulose and dissolving agent were mixed in a weight ratio of 5/95 and stirred at 400 rpm for 60 min at room temperature, followed by ultrasonic shaking for 5 min to prepare MC hydrogel. The mixture was placed in a freezer set at −18 °C and frozen for 12 h. After thawing, the mixture was stirred, ultrasonically shaken, and frozen again. The above freezing–thawing was repeated 3 times. After 3 freezing–thawing cycles, homogeneous cellulose solution was obtained.The method of cellulose hydrogel preparation used here followed a previous study . MC, CMC2-terminated WPU dispersion. The WPU dispersion was passed through a syringe filter with a 0.45 μm pore size (Millipore) to remove impurity. After filtering, the nonvolatile content, viscosity, pH value, and surface tension of the WPU dispersion were measured. The viscosity, pH value, and surface tension were measured with a Brookfield DV-E rotary viscometer, Suntex SP2100 , and FACE CBVP-A3  at 25 ± 2 °C.A prepolymerization process prepared WPU resin with PTMG, IPDI, DMPA, TEA, EDA, and water as the polyol, isocyanate, internal emulsifier, neutralizer, chain extender, and dispersed phase, respectively. Before synthesis, PTMG was placed in a 1000 mL four-neck glass reactor and dried in a vacuum oven to remove water. Then, DMPA and IPDI were added and reacted at 80 °C with a stirring speed of 200 rpm under a nitrogen atmosphere to undergo prepolymerization until the NCO content reached the theoretical value, which was measured according to ASTM-D2572 to form NCO-terminated prepolymers. At this stage, the reaction was conducted with molar ratios of 1.6/1 and 1.5/1 for NCO/OH and DMPA/PTMG, respectively. After cooling to 50 °C, TEA was added for neutralization for 30 min, followed by the addition of a calculated amount of deionized water dropwise under the stirring rate of 400 rpm. Finally, EDA was added to undergo chain extension reaction for 60 min, followed by continuous stirring at room temperature overnight to obtain a NHECH was used as the cross-linker to link two C6 alcohol groups in cellulose. The cellulose solution and ECH mixture with a molar ratio of epoxy group to hydroxyl group of 3/1 was stirred at room temperature for 20 min. Then, the calculated amount of WPU dispersion was added and stirred for 1 min to make the mixture uniform. The nonvolatile weight ratios of cellulose to WPU were set to 100/0, 95/5, 90/10, 85/15, and 80/20, and the formulation of composite hydrogels are shown in The compressive properties of the hydrogel were measured using a strength testing machine  at a load speed of 5 mm/min. Five repetitions were completed for each sample.1 and Wd1 are the weights of the hydrogel and aerogel, respectively.We placed the cellulose/WPU composite hydrogel in a refrigerator at −20 °C to freeze the water into an ice crystal. The frozen sample was dehydrated in a freeze dryer  for 4 days to form a dried cellulose/WPU composite aerogel. The gel fraction of the resin was calculated using the following formula:The microstructure of aerogel was observed with a tabletop electric microscope . The sample was fixed on the top of the sample stub with conductive double-sided tape, and an acceleration voltage of 15 kV was applied.d1) was immersed into distilled water, taken out, and weighed (Wr1) at 24 h and 48 h, and the reswelling ratio was calculated as follows:The cellulose/WPU composite aerogel (Wd2) in a freeze dryer, and the weight loss during the immersion process was calculated as follows:Then, the sample was dehydrated again  in a Np-value was 0.05.SPSS  was used for statistical analysis. One-way analysis of variance (ANOVA) with Scheffe’s test was used to calculate the statistical significance of results. The results are marked as English letters in the table and the bar graph, with different letters indicating statistical differences. The An amine-terminated WPU resin was prepared by a prepolymerization process. PTMG, IPDI, DMPA, TEA, and EDA were used as the raw material of polyol, isocyanate, internal emulsifier, neutralizer, and chain extender, respectively. The cellulose/WPU composite hydrogel was prepared by mixing WPU dispersion and cellulose solution and adding ECH as a crosslinking agent. Cellulose has no solubility in water and most organic solvents. The NaOH/urea solvent system can be employed to dissolve cellulose and thus vary the application . However2-terminated WPU dispersion. In preliminary experiments, it was found that if the cellulose solution, WPU dispersion, and ECH were mixed simultaneously, the reactivity of ECH to the amine group of the WPU dispersion is greater than the reactivity to the hydroxyl group of the cellulose. This phenomenon caused the molecular weight of the WPU resin to increase rapidly, forming agglomerates and separating them from the mixed system. To overcome this problem, the cellulose solution and ECH were mixed first and then left to react at room temperature for 20 min before adding the WPU resin. In this study, cellulose/WPU composite hydrogels were prepared by mixing cellulose solution and NH3 and 1.07 g/cm3. The gel fraction is the weight ratio of the hydrogel after dehydration to that before dehydration, and it can represent the weight percentage of the network structure in the hydrogel. The gel fraction of the hydrogel prepared with pure CMC is higher than those of pure MC and pure HEC. The introduction of WPU has no significant effect on the gel fraction of MC/WPU composite hydrogel, which is between 9.01% and 9.64%. However, after adding 5% WPU, the gel fraction of CMC/WPU composite hydrogel decreased significantly, but as the proportion of WPU increased, the gel fraction gradually increased. The gel fraction of hydrogel prepared by pure HEC is the lowest among the three celluloses, but the introduction of WPU can slightly increase the gel fraction.− groups [In this study, freeze-drying was used to remove water from a cellulose hydrogel with a volume of 35 mm × 30 mm × 20 mm to form a dry aerogel. We compare the appearance of composite aerogels made of different types of cellulose. As the proportion of WPU increases, the volume shrinkage of MC/WPU and HEC/WPU composite aerogels decreases, and a more complete block appearance structure can be maintained. This shows that the introduction of WPU can effectively preserve the pore structure of MC and HEC hydrogels and reduce structural shrinkage during the dehydration process. However, CMC/WPU composite aerogels with different WPU content present irregular shapes, and their volume shrinkage is greater than those of simple CMC aerogels.This study successfully introduced WPU resin into cellulose hydrogel to prepare cellulose/WPU composite hydrogel and aerogel. CMC/WPU composite hydrogels have swelling ratios higher than those of MC/WPU and HEC/WPU. The compression test shows that the structure of cellulose-based materials is the most important factor affecting the compressive stress of the composite hydrogel. The ones with MC as a raw material have higher compressive stress, followed by HEC and CMC. Cellulose/WPU composite hydrogel has good toughness and elastic recovery ability. The introduction of WPU can effectively maintain the pore structure of MC and HEC hydrogels and reduce volume shrinkage during dehydration. The microstructure shows that MC/WPU composite aerogel is porous, while HEC/WPU composite aerogel lacks an obvious pore structure. The cellulose/WPU composite aerogel with a weight ratio of 90/10 has the largest water absorption capacity, and the water absorption capacity values of the three celluloses are ranked as follows: CMC/WPU, HEC/WPU, and MC/WPU. The introduction of WPU resin can strengthen the network structure of the cellulose aerogel and reduce weight loss when immersed in water. The thermal degradation of cellulose/WPU composite aerogels mainly occurs at a temperature between 250 °C and 350 °C. The heat resistance of CMC aerogels can be improved by the introduction of WPU, and, at 90/10, the weight ratio of CMC/WPU has the best heat resistance.


In the present study, a tissue engineered SF scaffold containing simvastatin-loaded SFNPs were used to stimulate the regeneration of the defected bone.At first, the porous SF scaffold was prepared using freeze-drying. Then simvastatin-loaded SFNPs were made by dissolvation method and embedded in the SF scaffold. Afterwards, the scaffold and the NPs were characterized in terms of physicochemical properties and the ability to release the simvastatin small molecule.in vitro cell-based studies indicated that simvastatin-loaded SFNPs embedded in the scaffold had acceptable capacity to promote the proliferation and ALP production of osteoblast cells while inducing osteogenic matrix precipitation. The results exhibited that the SF scaffold had a porous structure suitable for releasing the small molecule and inducing the proliferation and attachment of osteoblast cells. SFNPs containing simvastatin had spherical morphology and were 174 ± 4 nm in size with -24.5 zeta potential. Simvastatin was also successfully encapsulated within the SFNPs with 68% encapsulation efficiency. Moreover, the small molecule revealed a sustained release profile from the NPs during 35 days. The results obtained from the The SF scaffold containing simvastatin-loaded SFNPs could have a good potential to be used as a bone tissue-engineered construct. More than 450,000 individuals suffer from bone defects in U.S. annually[1]. Bone tissue plays important roles in the human body, such as protecting the vital organs, blood production, preserving minerals, homeostasis, regulating blood pH, and producing mesenchymal stem cells and hematopoietic stems cell, and so forth. Bone defects are generally produced upon surgery, trauma, osteomyelitis, osteoarthritis, and bone fractures. Although growth factors are desirable components to induce osteogenesis, the high cost, low stability, low half-life, unwanted immunogenicity etc. restrict their usage in tissue engineering applications. It is assumed that small molecules could be potential alternatives for growth regeneration, cost-effectiveness, and higher stability. Small molecules are organic components with a molecular weight of less than 1,000 Dalton. The small size of these molecules facilitates their transit through the cell membrane phospholipid bilayer and triggers the signaling pathways inside the cell. The osteogenic small molecules are a group of materials that provoke the differentiation of multipotent mesenchymal stem cells into adult osteoblast cells[4-7]. Autologous grafts are the gold standards for treating bone injuries. However, the need for a second surgery, tissue removal, and restricted donor site limit the application of this type of strategy. Consequently, it is reasonable to use tissue engineering strategies as a potential alternative for autologous grafts to regenerate bone defects. A bone tissue engineered construct is usually composed of a biocompatible and biodegradable polymeric matrix containing various growth factors. The small molecules in the statin family are potential inhibitors that are widely used for treating hypercholesterolemia. Moreover, these molecules are capable to induce bone regeneration by stimulating the osteoblast cells and bone stromal cells through bone morphogenic protein signaling pathways. The slow release rate of statins from drug delivery systems also made them desirable molecules for treating osteoporosis. Simvastatin, lovastatin, atrovastattin, and rosuvastatin are some of the small molecules in the statin family and are capable of promoting osteogenesis[8-10]. To inhibit the off-target properties of statin on undesired tissues, it can be loaded in the targeted drug delivery carriers with the ability to release the cargo in the site of action. In recent years, various carriers, including microspheres, nanaospheres, nanocapsules, nanofibers, and so on, have been used for small molecule delivery. Among these systems, the nanoparticulate platforms have gained more attention due to higher stability, high surface to volume ratio, tunable size, and longer half-life. The polymeric NPs, whether synthetic or natural, are good candidates for small molecule delivery. However, it is necessary to choose a suitable polymeric construct according to the delivered molecule in terms of biocompatibility, biodegradability, solubility, controlled release behavior, mechanical strength, and chemical properties. Among different polymeric NPs, those synthesized from natural polymers are more utilized for bone regeneration due to less immunogenicity, acceptable biodegradability, and tunable processability[13]. In the present study, we used SF, as a potential natural polymer, for fabricating the bone scaffold[14-16]. To increase the ability of this scaffold for bone regeneration, we embedded the SFNP-containing simvastatin small molecules within the SF scaffold. It was hypothesized that this construct with great biocompatibility and biodegradability, high capacity for drug loading, good performance for cell endocytosis, controlled release behavior, and high half-life in blood circulation could enhance the proliferation and attachment of osteoblast cells in vitro.Recently, various osteogenic small molecules, such as dexamethasone, phenamil, oxysterol, purmor-phamine, fingolimod (FTY720), statin family, and bisphosphonates, have been used in bone tissue engineered constructsHerein, after the preparation of SF scaffold containing simvastatin-loaded SFNPs, the structure was characterized in terms of physico-chemical properties. The release profiles of simvastatin from both scaffold and SFNPs were also examined. Finally, the capability of the scaffold in provoking the osteoblast cells activities, such as proliferation, attachment, alkaline phosphate production, and matrix precipitation, were evaluated, as well. MaterialsSodium carbonate, lithium bromide, MTT, methanol, isopropanol, paraformaldehyde, simvastatin, and dialysis tube (cut-off 12 kDa) were purchased from Sigma-Aldrich (USA). DMEM, fetal bovine serum, and PBS were prepared from Gibco (USA). Rabbit osteoblast cells were obtained from the National Cell Bank of Iran .Preparation of SFNPs Bombyx mori silkworm silk fiber. For this purpose, the fibers were firstly dissolved in 0.02 M sodium carbonate for two consecutive 30 min, washed with PBS for three times, and dried at room temperature for 24 h. Secondly, the degummed fibers were dissolved in 9.3 M of lithium bromide at 60 °C for 4 h and then dialyzed using a dialysis tube against water for three days. At last, three types of SFNPs were prepared by adding ethanol, as a dissolving agent, to 2% SF solution. The NPs were then centrifuged at 35000 ×g for 10 min, and the participated NPs were freeze-dried for further use. The size and zeta potential of the prepared NPs were characterized using dynamic light scattering . The morphology of SFNPs was also analyzed by field emission scanning electron microscopy.Prior to SFNP preparation, the SF was extracted fromDrug loading efficacyTo encapsulate simvastatin into SFNPs, different concentrations of simvastatin  and a constant concentration of SFNPs (1%) were considered. For this purpose, the small molecule was dissolved in 1 mL of deionized water and then added to the SFNPs solution. The drug loading, encapsulation efficiency, and production yield were measured at 238 nm by spectroscopy using the following calculations: -1 wavelength. Moreover, to confirm the interaction between simvastatin and SFNPs, FTIR was performed at 400-4000 cmDrug release studyThe freeze-dried SFNPs were dissolved in 1 mL of PBS and transferred to a dialysis tube, immersed in 2 mL of PBS solution with pH 7.4 and kept at 37 °C. In each interval, the release medium was replaced with an equal amount of fresh PBS, and the cumulative release rate of simvastatin was measured at 238 nm using spectrophotometry. Preparation of SF scaffold containing simvastatin/ SFNPs To fabricate SF scaffold containing simvastatin/ SFNPs, 5 mg of the NPs were added and dispersed in 1 mL of degummed SF solution. The solution was then transferrred to a 24-well culture plate and preserved at -70 °C overnight. Afterwards, freeze-drying was applied for 48 h to fabricate the porous SF scaffold. Subsequently, the freeze-dried scaffold was crosslinked by immersion in methanol for two consecutive 10 min. The scaffold was then washed three times with PBS. The mechanical strength of the scaffold was examined in the pressure mode, triplicate. To assess the porosity of the scaffold, the freeze-dried SF scaffold was first immersed in ethanol and then removed after 10 min. Subsequently, the porosity percentage was measured according to the following formula:1 is the initial volume of ethanol, V2 is the volume of the ethanol after immersing the scaffold, and V3 is the residual volume of the ethanol after removing the scaffold from the solution.Where VScaffold biocompatibility2. After 24 h, the culture media was replaced with the extraction solutions of the scaffold and kept at 37 °C for another 24 h. The day after, the whole media was replaced with 0.5 mg/mL of MTT solution and placed in an incubator for 4 h. Finally, the participated formazan was dissolved by isopropanol, and the absorbance was read at 570 nm using an ELISA reader. Three groups were considered for MTT assay: (1) TPS, (2) bulk SF scaffold, and (3) SF scaffold containing simvastatin/ SFNPs. The biocompatibility of the scaffold was evaluated using an indirect test according to ISO 10993-5 standard. To this end, the scaffolds with an identified surface area were immersed in 1 mL of culture medium and kept for cell viability test for 3, 7, and 14 days. Then rabbit osteoblast cells were cultured in a 24-well culture plate at 37 °C and 5% COALP activityThe rate of ALP production by the osteoblast cells was measured by a commercial ALP kit  according to manufacturer's protocol. Briefly, about 5,000 osteoblast cells were cultured in a 24-well culture plate for 24 h. The culture medium was replaced with the extraction solutions of the scaffolds and kept in an incubator for three days. Then the supernatant of the rabbit osteoblast cells (containing 10 μL of cells) was added to the ALP kit reagent , and the absorbance was read at 405 nm using an ELISA reader . Alizarin red stainingTo confirm the ability of rabbit osteoblast cells for mineralization and matrix calcification next to the SF scaffold, Alizarin red staining was performed. Firstly, 5,000 osteoblast cells were cultured in a 24-well culture plate. After 24 h, the culture medium was replaced with the extraction solutions of the scaffold for three days. Afterwards, the cells were washed with 0.1 M of NaCl and fixed in 1% paraformaldehyde (4 v/v%) for 30 min and stained with Alizarin red at room temperature. The stained regions were evaluated using light microscopy, and the results were reported, qualitatively. Statistical analysisp values less than 0.05 were considered statistically significant.Statistical analysis was performed by using one-way ANOVA with SPSS 16.0 . Size and zeta potential of SFNPsBased on the dynamic light scattering data, the size of SFNPs was 146 nm with PDI 0.2  before dSurface morphology of SFNPsSEM micrographs showed that SFNPs had spherical morphology without accumulation due to the repulsion of negative charges at the surface of NPs. This phenomenon resulted in the uniform distribution and stability of SFNPs. As can be seen in Structural analysis of SFNPs-1, 1518 cm-1, and 1630 cm-1, respectively (-1 and 3749 cm-1 (O−H stretch vibration), as well as at 1730 cm-1, and 1164 cm-1 and 1066 cm-1 . However, after loading simvastatin on SFNPs, no significant changes were observed in the FITR spectra of SFNPs because most of the drug molecules were encapsulated within the NPs and a slight portion of the drug was loaded only on the SFNPs . Moreover, the ALP production on day 14 was more than days three and day seven . This trend was also observed on day 14 .


Our findings support the idea that EBV is related to laryngeal carcinoma. However, further studies are needed before recognizing a definitive etiological role of EBV in the development and/or progression of laryngeal carcinomas.Over the past few decades, several publications have investigated the role of Epstein-Barr virus (EBV) in head and neck squamous cell carcinomas, and an increasing number of them have shown its presence in laryngeal tumors. The purpose of this meta-analysis was to evaluate the association of EBV with laryngeal carcinoma. The search was carried out in two databases, Scopus and PubMed, using the following terms: “Epstein-Barr virus” and “laryngeal carcinoma”. A total of 187 records were found, of which 31 were selected for meeting the inclusion and exclusion criteria. The meta-analysis yielded an overall pooled prevalence of 43.72% : 34.35–53.08). Studies carried out in Europe and Eurasia had slightly higher pooled prevalence than other subgroups, while the prevalence of studies performed in developed countries was higher than in developing countries (46.37% vs. 34.02%). Furthermore, laryngeal carcinoma occurred almost three times as often among EBV-infected individuals compared to those without EBV infection (odds ratio = 2.86 (95% CI: 1.18–6.90); Begg’s test,  According to the global estimates from the International Agency for Research on Cancer (IARC), for 2018, there were 177,422 new cases and 94,771 deaths due to cancers of the larynx. Both values  represented about 1.0% of all 36 cancers analyzed [Although it is well known that tobacco smoking and alcohol consumption are the major risk factors for the development of laryngeal SCC, infectious agents may also be implicated in the pathophysiology of some cases ,4. In thAdditionally, various genotypes of human papillomavirus (HPV) have been found in samples of laryngeal SCC . Thus, tBřicháček et al.  were theIn the present study, we performed a systematic review with meta-analysis of articles available in the international literature on the association of EBV with carcinomas of the larynx, in order to answer the following research question: Are EBV-infected individuals at increased risk for the development and/or progression of laryngeal carcinoma? Characteristics of the studies, such as reported prevalence, techniques used, detection targets, and other relevant aspects for understanding of the relationship between EBV and laryngeal carcinomas, were evaluated.n = 8) [n = 5) [n = 3) [n = 1) [n = 1) [n = 1) [The 31 systematically reviewed studies were carried out in 19 different countries in Europe n = 12) [ 12,13,1,18,19,20n = 8) ,26,27,28 [n = 5) ,30,31,32 [n = 3) ,5,33, Oc [n = 1) , South A [n = 1)  Table 1n = 8 [n n = 16) [n = 14) [n = 10) [The studies have detected EBV by PCR-based techniques n = 16) ,32,33,34 ,32,33,3[n = 14) ,31,32,35[n = 10) ,29,30,35[n = 10) ,28.Some articles also explored the presence of other viruses. In order to investigate the possible association between viral (co)infection and a laryngeal carcinogenesis, these data were also considered in the present systematic review and summarized in p < 0.001). p < 0.001). Subgroups that used the in situ hybridization and PCR-based technique yielded the following pooled prevalence: 49.58%  and 39.31% , respectively.Collectively, the studies included in the present review gathered 1292 distinct specimens from patients with laryngeal carcinoma. Twenty-nine studies could be submitted to meta-analysis, of which eight studies presented a prevalence of 0 or 100% and were automatically excluded from the analysis. The overall pooled prevalence was 43.72% .p < 0.001) was higher than in developing countries.Studies carried out in Europe and Eurasia had slightly higher pooled prevalence than other subgroups, whereas the prevalence of studies performed in developed countries ; in comparison, for the control group composed of non-malignant lesions, the chance of developing LC in EBV-positive individuals was nearly twice as common as in the non-infected group (OR = 1.94 (1.18–3.21); p = 0.299). The results of the p-value and I-squared statistics in the latter analysis revealed low heterogeneity (I2 < 50% and p > 0.05), therefore indicating the fixed-effects model as the most suitable, and both models had similar odds ratio results . The overall result showed values closely related to those of the second subgroup (OR = 2.18 (95% CI: 1.33–3.59); p = 0.007). p = 0.843) and Egger’s test (p = 0.866). No evidence of publication bias was observed.In our sample, 15 case-control studies were identified and used in the odds ratio analysis ,30,33,35The hypothesis that viral infections are associated with the development of human cancers has been studied since the early 1960s, when the first human oncovirus was discovered in Burkitt’s lymphoma: the Epstein-Barr virus . In 1973One year later, the first study evaluating the involvement of EBV in laryngeal carcinoma was published; however, the authors failed to show any significant antibody titer differences between carcinoma and control groups, leading them to suggest that EBV is not etiologically related to LC . PerhapsIn the same year, Callaghan et al.  found thThereafter, other studies have sought to assess the relationship between EBV and laryngeal carcinoma, and the number of publications on this subject gradually increased in the following decades, but in a smaller proportion compared to other EBV-related tumors . As shown in Among the studies included, some were not able to detect EBV in their samples, either by ISH ,22,26,31It is worth mentioning that studies such as Yang et al.  and de ORegarding the studies employing PCR-based methods, most demonstrated positivity for EBV with an average prevalence around 50%. However, these results face the same dilemma observed in carcinomas from other anatomical sites, that is, the real meaning of the expressive PCR positivities given the impossibility of determining the precise source of viral DNA, whether from tumor or non-malignant cells . In this regard, when PCR-based methods are used for EBV detection, the results must be considered with caution because it has been estimated that EBV latently infected B-cells may be found in circulating cells in healthy individuals, and thus false-positive results must be considered ,3. In a In the present review, even without applying a time limitation filter in the searches, the number of articles retrieved evaluating the involvement of EBV in LC is still small. Moreover, many studies enrolled small samples, some applied indirect EBV detection methods , while only a few were able to demonstrate the viral genome or its products (transcripts or proteins) in tumor cells, and there is a lack of information on the distribution patterns of staining. In addition, there are some relevant confounding variables, such as alcohol consumption and tobacco smoking that are traditional risk factors for HNSCC, as well as other oncogenic viruses that are also found in this site, sometimes in coinfections, which compromise the assessment of the role of EBV in the initiation and/or progression of laryngeal carcinomas. In this context, Mulder et al.  were unaTherefore, some authors recognize that the role of EBV in the pathogenesis of laryngeal carcinoma is still unclear ,25,32,33In a Turkish study, the identification of EBV DNA in 50% of patients with laryngeal carcinoma and in 41.2% of the vocal cord nodules indicates, according to the authors, that this agent does not seem to be directly associated with the pathogenesis of LC, but suggests a causal relationship involving EBV and proliferative diseases in the larynx . In the Three studies could not find any significant difference between EBV DNA positivity and smoking habits ,32,33. TInterestingly, two studies failed to detect EBV by EBER-ISH in laryngeal lymphoepithelial carcinomas, denoting the peculiarity of this rare type of neoplasm ,31. AcuñDespite the controversy, some authors consider that EBV plays a role in the development of LC ,23,25,27Although the possibility of false-negative cases is admitted in those studies investigating the presence of EBV in samples taken from FFPET, since formalin is a known inhibitor for PCR ,27, in oInterestingly, the developed countries demonstrated higher pooled prevalence than developing ones. Although there is little information in this regard, one could argue that the difference observed between developing and developed countries might be related to the period of primary EBV infection. According to Ali et al. , due to In a recent case-control study, the EBV DNA positivity detected by qPCR was significantly higher in LC group (52% vs. 20%), showing that individuals with laryngeal EBV DNA positivity had a greater risk of developing LC (unadjusted OR = 4.3). Also, EBV DNA presence was positively correlated with BCL-2 expression (an anti-apoptotic protein) . Althougp = 0.004).Despite the debate on the high sensitivity of PCR-based methods which may incur false-positive results, in a multivariate analysis performed by Lee et al. , EBV DNARegarding histological approaches, although evidence on the location of EBV is scarce, some studies have shown distinct nuclear staining by ISH in tumor cells of laryngeal SCC ,28,35. IThe cases in which EBV DNA was found but viral protein was not detected ,32 may iSome experiments have indicated that EBV infection may affect the immune response in the context of laryngeal carcinogenesis. For instance, the findings of Klatka et al.  point toThe importance of studies focusing on the impact of coinfections on the malignant transformation of the laryngeal epithelium have also been highlight, which may contribute to clarifying their association with the carcinogenic process and to guide prophylactic strategies ,17,18. IIn our analysis, five studies reported EBV and HPV coinfections ,15,17,18Although the role of EBV/HPV coinfection has been hypothesized, the coexistence of both viruses has not been proven to have an unfavorable implication in the prognosis of LC patients . In addiOther viruses detected in LC include HSV-1, CMV, HHV-8, HIV, BKV, and MCPV ,17,18,35In general, the limitations of the present study were related to the use of only two databases and the inclusion of articles exclusively published in English that may have restricted the number of records found. Moreover, as studies on EBV in laryngeal carcinoma are less common, a time period was not established in the searches, resulting in the selection of a greater number of articles using indirect methods for EBV detection, which along with the small sample sizes observed in some works and diversity of techniques employed are likely the main causes of the heterogeneity verified in our meta-analysis. Finally, the review being centered on observational studies, due the nature of the investigated subject, may have downgraded the strength of the current evidence.To investigate the association of EBV with laryngeal carcinoma, we conducted a systematic review with a meta-analysis of publications that were retrieved from the electronic scientific databases PubMed and Scopus, using the following search terms: “Epstein-Barr virus” and “laryngeal carcinoma”. In the PubMed database, the search was set to include all fields, while in Scopus the search was conducted based on the title, abstract, and keywords. In view of the limited number of articles available, no filter was applied for the time period; for this reason, the search covered a relatively long period (November 1973 to 3 September 2021).The manuscripts were selected primarily through the analysis of titles and abstracts. The publications were reviewed independently by three authors and submitted to the following inclusion criteria: (i) articles written in English; (ii) articles addressing the association between carcinoma of the larynx and EBV; and (iii) original studies. Publications were excluded which (i) employed no EBV detection method (direct or indirect); (ii) were not classified as original research ; (iii) involved only squamous cell carcinomas from other locations in the upper aerodigestive tract and did not include any laryngeal cases, or combined the LC cases with tumors from other anatomical sites without presenting the values separately; (iv) were based on animal samples or models; or (v) were repeated documents. Any disagreement during the selection of records was resolved by the senior author.Initially, we found 109 records in PubMed and 78 in Scopus. Fifty-nine duplicate records were excluded. After filtering for language and type of study and evaluating the connection of the content with the proposed aim, 31 articles were included in the systematic review . Full-te2) and the Q statistics, with the level of statistical significance set at 5% (p < 0.05). The EBV pooled prevalence was initially estimated with a fixed-effects model. However, due to the significant level of heterogeneity observed, we conducted a second analysis using the random-effects model of Der Simonian and Laird [The statistical analysis was performed with the software Stata, version 12.0 , using the following commands: “metan”, “metafunnel”, and “metabias” . First, nd Laird . StudiesTo our knowledge, the present study represents the first systematic review with meta-analysis of the literature on the association of EBV with carcinoma of the larynx, with relevant evidence such as the expressive pooled EBV prevalence of 43.7% in samples from LC patients and the fact that EBV-infected individuals are 2.86 times more likely to develop laryngeal carcinoma. Despite the controversies, our findings indicate a possible role for EBV as a risk (co)factor in the development and/or progression of LC. Moreover, these tumors seem to share similarities with other HNSCCs, as well as with anogenital and breast carcinomas in which EBV can be found alone or in coinfection with high-risk genotypes of HPV, strengthening the thesis that both oncoviruses can cooperate in the epithelial transformation. There may also be an influence of EBV in the immunological response in the context of laryngeal carcinoma, providing an appropriate environment for the establishment of the LC, by promoting tumor growth and by suppressing anti-tumor responses. On the other hand, unlike other EBV-related carcinomas, there are still few studies in the literature dealing with this topic in the larynx; consequently, the location and distribution of EBV in the tissue remain controversial and there is a lack of information about its transcriptional activity, and, in turn, about the expression pattern of viral genes, whose available data only allow us to assume that EBERs are expressed in these tumors. Hence, further studies are needed in order to elucidate the inconsistencies. In this sense, large and standardized case-control and cohort prospective studies, focusing on cases unrelated to confounding factors for the upper aerodigestive tract, combining different techniques and targets for EBV detection, may contribute to the understanding of the role played in this context. Also, in vitro studies are particularly important to investigate the effects of EBV on laryngeal carcinoma cell lines and possible underlying tumor mechanisms. Taken together, the present findings suggest that EBV may be associated with laryngeal carcinomas and contribute to expanding the knowledge on this oncovirus, in addition to encouraging new studies which, ultimately, may improve the clinical management and prevention of LC cases.


Family planning (FP) is a key intervention for preventing unplanned pregnancies, unsafe abortions, and maternal death. Involvement of both women and their partners promotes contraceptive acceptance, uptake and continuation, couple communication and gender-equitable attitude. Partner involvement is a key strategy for addressing about 17.5% of the unmet needs in FP in Kenya. This study assessed the prevalence and factors associated with covert contraceptive use (CCU) in Kenya.We used data from the sixth and seventh rounds of the performance monitoring for accountability surveys. We defined CCU as “the use of contraceptives without a partner’s knowledge”. We used frequencies and percentages to describe the sample characteristics and the prevalence of CCU and assessed the associated factors using bivariate and multivariable logistic regressions.The prevalence of CCU was 12.2% (95% CI: 10.4–14.2%); highest among uneducated (22.3%) poorest (18.2%) and 35–49 years-old (12.8%) women. Injectables (53.3%) and implants (34.6%) were the commonest methods among women who practice CCU. In the bivariate analysis, Siaya county, rural residence, education, wealth, and age at sexual debut were associated with CCU. On adjusting for covariates, the odds of CCU were increased among uneducated women , women with primary education  and those from the poorest , poorer , and middle  household wealth quintiles and were reduced among those with 2–3  and ≥ 4 children . Age at sexual debut  reduced the odds of CCU.About one in 10 married women in Kenya use contraceptives covertly, with injectables and implants being the preferred methods. Our study highlights a gap in partner involvement in FP and calls for efforts to strengthen their involvement to increase contraceptive use in Kenya while acknowledging women’s right to make independent choices. Kenya has a high fertility rate of 3.9 births per woman , a teenaMost FP interventions have been targeted towards women, who are the primary users of contraceptives. The lack of involvement of male partners results in opposition to self or partner’s FP use –6 due toMoreover, women have resorted to covert contraceptive use (CCU) due to fear of reprisal from the partner , religioCCU is common in male-dominated settings where women lack the autonomy to make choices on their health . StudiesCovert contraceptive use is widespread in sub-Saharan Africa, with a prevalence of 2.6–53% , 28, 29.While covert use of contraceptives may contribute to the overall increase in modern contraceptive prevalence rate in Kenya, which currently stands at 58.1% , it coulWe utilised data from sixth  and sevethe use of contraceptives without a male partner’s knowledge” [Does your partner/husband know that you are using family planning?” among women currently using FP and in-a-union, for which they responded either ‘yes’ or ‘no’.CCU, the outcome variable, was defined as “owledge” . It was Nairobi, Bungoma, Kericho, Kiambu, Kilifi, Kitui, Nandi, Nyamira, Siaya, Kakamega and West Pokot) while the locality of residence was either rural or urban, based on the classification by the KNBS master sampling framework [no formal, primary, secondary and tertiary) [poorest, poorer, middle, richer, richest) were computed based on wealth index generated using principal component analysis of the household assets, walls, flooring and roofing materials and type of water access and sanitation facilities [How many times have you given birth?” and the response recoded as 0, 1, 2–3 and 4+ [How old were you when you first had sexual intercourse?” and the response recorded in years [yes’, ‘no’ or ‘infertile’ [The independent variables were selected based on a review of the literature on FP and the availability of the variables in the dataset. They included the county of residence, locality of residence, age in years, education levels, wealth quintiles, parity, desire for more children and age at sexual debut in years. The county of residence included the 11 counties sampled in the survey  , 27, 38.cilities . Parity 3 and 4+ . Women’sin years . Women wfertile’ .p-value = 0.05.We described the sample characteristics and the prevalence of CCU using frequencies and percentages. Factors associated with CCU were assessed using bivariate and multivariable logistic regressions. All variables in the bivariate analysis were included in the multivariable analysis. Stata 13.0 was used for analyses , which wOf the 11,753 sampled women, 3943 were in a union and currently using contraceptives. We included 3892 women after excluding 49 women (1.3%) who did not respond to the question on their partner’s knowledge of their contraceptive use . It was highest among uneducated (22.8%), poorest (18.1%) and older  women; and among those living in rural areas (13.7%) and neither had children (20.8%) nor wanted for more children (13.3%). Among the counties, Siaya (21.9%), Kericho (16.8%) and Kakamega (12.6%) counties had the highest CCU prevalence (Table Siaya county, rural residence, education, wealth, and age at sexual debut were associated with CCU in the bivariate analysis. In the multivariable logistic regression, counties, rural residence, education, wealth, parity, and age at sexual debut were associated with CCU Table . UneducaThe present study found a 12.2% prevalence of CCU among married women in Kenya. The prevalence was high among older, uneducated, poorest, and rural women, and among women who neither had children nor wanted for more children. The commonest methods of contraceptives used covertly were injectables and implants. Education levels, wealth, county of residence, rural residence, parity, and age at sexual debut were associated with CCU.The prevalence of CCU in SSA ranges from 2.6 to 20.2% . In our Similar to previous studies in Ethiopia , Ghana , Kenya , 24 and Girls’ education delays early marriages, reduces the age at sexual debut and improves girls’ and women’s self-esteem , which iWe also found that women in rural areas were more likely to practice CCU compared to those in urban areas, though not statistically significant. Married urban women may be more involved in decision making on children and FP due to their high level of knowledge on contraceptive and gender-equitable attitude . On the Our study is one of the first to explore the practice of CCU in Kenya. We used the most recent nationally representative cross-sectional data from 11 out of the 47 counties of Kenya, which makes our findings generalizable to the country. The major strength of this study is that it focuses on women-in-a-union and excludes women not-in-a-union who we could not ascertain whether they had partners. Our study findings are, therefore, important for informing immediate FP policies and interventions that focus on married couples. However, based on the cross-sectional nature of the data we could not infer causation. Also, some key determinants of CCU such as duration of the marriage, years of schooling, decision making and exposure to FP messages in the media were not collected. Regardless, our findings are comparable to those of similar settings and provide the most recent preliminary evidence on the extent and factors associated with CCU in Kenya.Our study shows that about one in 10 married women in Kenya use contraceptive covertly, with injectables and implants being the preferred methods. County of residence, education levels and rural residence were positively associated with CCU while wealth parity and age at sexual debut were negatively associated with CCU. CCU could reflect both increased or decreased women autonomy and independence in decision making on their health. While it is important to promote open contraceptive use among married couples, women should be supported to use contraceptives in whichever way they prefer. Importantly, FP programmes should promote couple’s communication and foster male involvement in FP to improve overall contraceptive use. Further research is needed to explore the reasons for CCU in Kenya, the role of unmarried women in CCU studies and whether unmarried women have the moral obligation to communicate their use of FP to their sexual partners.


Diabetes is present in 10.5% of the US population and accounts for 14.3% of all office-based physician visits made by adults. Despite this established office-based approach, the disease and its adverse outcomes including glycemic control and clinical events tend to worsen over time. Available home technology now provides accurate, reliable data that can be transmitted directly to the electronic medical record.This study aims to evaluate the impact of a virtual, home-based diabetes management program on clinical measures of diabetes control compared to usual care.1c (HbA1c), creatinine, estimated glomerular filtration rate, and insulin use in a usual care group after 1 year. Digital medicine patients completed questionnaires online, received medication management and lifestyle recommendations from a clinical pharmacist or advanced practice provider and a health coach, and were asked to submit blood glucose readings using a commercially available Bluetooth-enabled glucose meter that transmitted data directly to the electronic medical record.We evaluated glycemic control and other diabetes-related measures after 1 year in 763 patients with type 2 diabetes enrolled into a home-based digital medicine diabetes program and compared them to 794 patients matched for age, sex, race, BMI, hemoglobin A1c  or changes in the proportion of patients with HbA1c≥9.0 . Digital medicine patients demonstrated improvements in HbA1c  and significant changes in the proportion of patients with HbA1c≥9.0 , diabetes distress , and hypoglycemic episodes .After 1 year, usual care patients demonstrated no significant changes in HbAA digital diabetes program is associated with significant improvement in glycemic control and other diabetes measures. The use of a virtual health intervention using connected devices was widely accepted across a broad range of ethnic diversity, ages, and levels of health literacy. In 2019 greater .1c, and less than 15% meet all three targets of glycemic control, blood pressure, and low-density lipoprotein (LDL) cholesterol [Although progress has been made to improve risk factors for microvascular and macrovascular disease in diabetes, approximately half of individuals with diabetes do not meet individualized targets for HbAlesterol -10. Morelesterol .Several factors account for these poor outcomes, including the use of suboptimal numbers or doses of medications, therapeutic inertia, lack of patient engagement, and limited resources and time to educate and provide lifestyle recommendations ,13. AlthHome blood glucose monitoring with direct transmission of data to the medical record addresses several limitations of traditional office-based care, including a larger sample of biologic data and an ability to take more timely action including therapy modifications -19. CurrWe sought to evaluate the effectiveness of a remote, home-based telemonitoring program in a clinical setting using commercially available technologies on glycemic control in adults with type 2 diabetes (T2D).1c, lipid levels, creatinine, estimated glomerular filtration rate (eGFR), thyroid function tests, urine protein, and BMI as well as completion of a retinal examination. This data was used to create a patient phenotype that assisted in the design of the intervention process. As an example, a poorly controlled individual with low health literacy and financial stress would be differentiated from an individual with high patient activation and reduced physical activity. Digital glucose meters were obtained, and initial training and setup was provided at the Ochsner O Bar, a patient-facing service that provides information, training, and tech support for patients interested in apps, wearables, and connected home devices [As part of a quality improvement initiative, adult patients with the diagnosis of T2D at the Ochsner Health System  were offered enrollment into a digital diabetes program by their physician during an office encounter or through an offer letter. Patients were required to possess a smartphone, purchase a Bluetooth connected glucose meter  that had direct access to the electronic medical record , and have an active account in the patient portal (MyChart). If patients did not have an active patient portal account, they were given the opportunity and online assistance to sign up for one. Program details, questionnaires, and electronic consent to participate took place online through MyChart. Questionnaires assessed factors related to diabetes and chronic disease management, including diet, physical activity, depression, medication adherence, patient activation, health literacy, and social circumstances . Diabetes-related emotional distress was assessed using the Diabetes Distress Scale ,21. Heal devices . All blo1c, creatinine, eGFR, and insulin use, and were followed as a usual care group over time.A second control group of patients who met all eligibility criteria but whose physician was either not participating in the program or did not choose to enroll patients were followed. Of these, 794 patients were propensity matched to the digital medicine group according to age, sex, race, BMI, HbADoctoral pharmacists, advanced practice providers (APPs) including nurse practitioners and physician assistants, and health coaches participated in the intervention that included education, lifestyle recommendations , and medication management as per diabetes guidelines ,23. EachEach doctoral pharmacist, APP, and health coach received training in diabetes management and education as well as use of the custom tools within the EMR created to facilitate optimal management ,19. DoctPharmacists and APPs contacted patients by phone and discussed screening results and treatment options for achieving glycemic control. Patients were encouraged to actively participate in their diabetes management and collaborated with the doctoral pharmacist or APP to cocreate the treatment plan by choosing among various lifestyle and medication recommendations . CurrentPatients with medication affordability issues were, as much as possible, switched to generics or less expensive combination agents and, when appropriate and feasible, enrolled in medication assistance programs. Those with medication adherence issues were provided educational materials, pill reminder apps and resources, and a simplified medication regimen when possible.1c and upcoming health maintenance metrics as well as lifestyle tips based on their screening phenotype. Physicians also received monthly reports on their patient’s progress including blood glucose control and health maintenance metrics. Incoming glucose data was analyzed via internally developed algorithms as to its validity and directional change, and alerts were established to highlight which patients needed what intervention and when. Individualized targets for HbA1c were created based on current guideline recommendations [Patients received monthly reports by mail and electronically detailing their progress, including HbAndations .1c measurements, retinal imaging for retinopathy, and screening for diabetic kidney disease).The primary outcome was the proportion of patients achieving their glycemic control. Secondary outcomes included incidence of hypoglycemia and achievement of health maintenance measures , differences in group means, 95% CIs, and P values. To evaluate changes in medications from baseline to 12 months, a logistic model for repeated measures was constructed [P values. Because no patients in the digital medicine program were treated with alpha glucosidase inhibitors, this medication class was not included in the analysis. A significance level of .05 was used for all statistical tests. Analyses of outcomes were carried out using SAS version 9.4 for Windows (SAS Institute).To assess association between the digital medicine program for diabetes and patient outcomes, general linear models were used . Distribstructed . The mod1c target, while 107 patients (14%) had HbA1c values greater than or equal to 9.0% (range 9.0-13.9).We evaluated all patients at baseline and again following 1 year. Prior to enrollment in the digital diabetes program, patients were under the care of their primary care clinician for their diabetes for an average of 5.2 years, averaging 2.8 visits per year. Of the 763 patients at baseline, 328 (43%) patients had not achieved their goal HbA1c, creatinine, eGFR, and use of insulin. There were, however, lower levels of total and high-density lipoprotein (HDL) cholesterol as well as higher systolic and diastolic blood pressure in digital medicine patients. It is noteworthy that digital medicine patients’ age ranged from 31 to 98 years, with 28% (n=215) of enrollees 70 years or older.The baseline characteristics of the digital diabetes (n=763) and the propensity matched usual care groups (n=794) are outlined in P<.001). Finally, a net promotor score (likelihood to recommend the program to a friend or colleague) survey was collected yielding 385 responses (50% response rate), generating a score of 83 (range –100 to 100).Additional characteristics were uniquely obtained in digital medicine patients. At entry, 168 of the 763 (22%) patients exhibited symptoms of depression, 160 (21%) patients described financial difficulty in paying for medication, and 122 (16%) patients had low levels of health literacy. Basic technology skills were assessed at entry, of which 69 (9%) patients scored as deficient ,32. Mode1c, fasting glucose, or blood pressure but did show improvements in total cholesterol and BMI.1c, lipids, BMI, and diastolic blood pressure, which took place without changes in lipid or antihypertensive therapy. Digital medicine management yielded a 57% reduction in the percent of patients whose HbA1c≥9.0% , whereas no statistically significant change was observed in usual care patients  in total hypoglycemic episodes with a commensurate 82% reduction in frequency to 0.15 episodes per patient per month (P<.001).Baseline and 1-year hypoglycemic episodes were recorded in digital medicine patients . A total1c, HDL and LDL cholesterol, and diastolic blood pressure in the digital medicine managed patients compared to usual care.The average change in these metrics per patient are described in 1c levels, attainment of goal HbA1c, lipid levels, diabetes distress, and annual health maintenance adherence compared to usual care. Second, hypoglycemia is frequently present and is often unrecognized in individuals with diabetes managed in clinical practice; the severity and frequency of hypoglycemic events can be markedly reduced with timely knowledge and intervention through real-time capture of home self-monitoring of blood glucose [There are several important findings from this investigation. First, the digital health monitoring and intervention program significantly improved HbA glucose ,34. FinaThe standard management of diabetes in clinical practice has significant system and process limitations including limited episodic interactions with patients, access to a small fraction of glucose data, a reduced ability to evaluate patient comprehension and needs, and a brief amount of time to educate and reinforce essential health-promoting behaviors ,33-36. P1c targets, reduction of hypoglycemia, reducing diabetes distress, and completing necessary health maintenance. These improvements are in contrast to outcomes that often occur when people with diabetes are seen only 2 to 3 times a year, resulting in persistence of hyperglycemia or hypoglycemia over prolonged periods of time [1c remained unchanged after 1 year. Moreover, after an average of 5 years under the care of their primary care physician, 41% (n=314) of patients were discovered to have hypoglycemic events each month, of which 6% (n=46) were level 3. Finally, the digital intervention was provided to a diabetes population with a broad range of ages (range 31-98 years), races, and health literacy, and it overcame many potential barriers to care, including access.We have demonstrated that redesigning care delivery using a digital health program can substantially improve multiple metrics important in caring for patients with T2D, namely, achievement of HbA of time ,34. ThisThere are several factors worth describing in our care delivery redesign. First, the clinical team of doctoral pharmacists and APPs prescribed medications based on current guideline recommendations, which contrasts the high rate of care variation observed in routine practice -42. SecoIt is noteworthy that costs of care were not assessed, and future studies will be needed to determine the financial impact of this digital intervention.There are, however, several limitations to this study. First, this was a single-center study with a 1-year follow-up occurring in an integrated health care delivery system and involved a relatively modest number of individuals whose average glycemic, blood pressure, and lipid control were not markedly abnormal. Despite the advantages of Bluetooth-enabled glucose meters, it is likely that the use of continuous glucose monitoring (CGM) would have identified a greater number of hypoglycemic events. With recent improvements in CGM performance and availability, it is likely this technology will be offered to appropriate participants in the future. Second, patients were not prospectively randomized into intervention and usual care groups. Finally, only patients who possessed a smartphone were eligible to enroll, which raises issues about education, socioeconomic, and motivational biases. However, the mean age of our population was 63 years, and on screening, 9% (n=69) lacked common technology skills, suggesting that our cohort was not biased toward a younger, more technically competent population.In summary, a team-based digital health program that incorporates a higher frequency of real-time glucose data and touchpoints with the clinical team is an effective modality for delivering diabetes management, outperforming traditional office-based care. Although the bulk of our results were obtained prior to the COVID-19 pandemic, this mode of care provides unique value in this setting.


All were subjected to in-situ zymography. Dentin slices were, subsequently, obtained, covered with fluorescein-conjugated gelatin, and examined with confocal laser-scanning microscopy. The fluorescence intensity was quantified and statistically analyzed using one-way ANOVA and Bonferroni tests (α = 0.05). Furthermore, gelatin zymography was performed on protein extracts obtained from dentin powder (N = 8 teeth), treated with hydrogen peroxide or carbamide peroxide, with different exposure times . The results of the in-situ zymography showed no statistical differences between the bleached specimens and the control group, with a medium level of gelatinolytic activity expressed in the dentin tubules. The results of gelatin zymography showed an increased expression of pro-MMP-9 in carbamide peroxide groups. The expression of pro-MMP-2 decreased in all the experimental groups. The bleaching treatments performed on the enamel of sound teeth do not influence dentinal enzymatic activity. However, when unprotected dentin tissue is bleached, matrix metalloproteinases are more expressed, particularly when carbamide peroxide is used, proportional to the exposure time.This study aimed to investigate matrix metalloproteinase (MMP) activity in human dentin using in-situ and gelatin zymography, after at-home and in-office bleaching, related to their clinical exposure times. Dentin specimens ( Esthetics has gained growing recognition in dentistry, reflecting an increasing demand of patients who consider the smile a factor of great relevance ,2. One o−) and singlet oxygen (1O2) [Currently, two professionally supervised techniques are commonly reported: the in-office and the at-home bleaching. The most commonly used active ingredients are hydrogen peroxide (HP) and carbamide peroxide (CP), applied in different concentrations, depending on the application method ,6. Bleacen (1O2) . These cen (1O2) ,5. Clinien (1O2) .2+/Zn2+-dependent endopeptidases of the extracellular matrix, involved in numerous physiological and pathological processes. It is assumed that these enzymes have active roles in tooth formation, and, after the mineralization of dentin, they remain “fossilized” and, hence, inactive. The MMPs secreted by the odontoblasts, however, have a lifelong role in the response of tooth tissues to inflammation or other exogenous factors. Furthermore, the inactive dentinal MMPs can be reactivated by different processes, such as caries and dental restorative procedures. Actually, various dental materials  were proven to cause an increase in the activity of the MMPs [The dentin matrix is a complex network of fibrillar and globular structures that form the organic scaffold of dentin. Matrix metalloproteinases (MMPs) and cysteine cathepsins (CC) are Cathe MMPs ,12,13,14the MMPs ,17,18,19the MMPs . HoweverAccordingly, the objective of this study was to evaluate, qualitatively and quantitatively, the influence of both at-home and in-office bleaching treatments on dentinal enzymatic activity using in-situ zymography and gelatin zymography. The null hypotheses in this study were: (1) in-office and at-home bleaching through the enamel does not influence dentinal enzymatic activities; and (2) the treatment of dentin powder with bleaching agents at different exposure times does not influence the expression of MMP-2 and -9.p > 0.05; ANOVA). The intensity of fluorescence, corresponding to the level of enzymatic activity within the dentin structure, can be observed in The enzymatic activity  of the investigated groups is described in The zymograms of gelatinolytic activity are shown in The present study evaluated the effects of different bleaching protocols on dentinal enzymatic activity using, for the first time, both in-situ zymography and gelatin zymography. Since the results demonstrated that there were no differences in the dentinal enzymatic activity between the control and experimental groups when the bleaching was applied through the enamel, the first null hypothesis was accepted. On the other hand, the gelatin zymography of the dentin powder showed differences in the expression of the MMPs between the groups, warranting the rejection of the second hypothesis.The in-situ zymography was performed, aiming to investigate the more clinical approach, applying the bleaching treatment onto the enamel and evaluating the effects on dentin tissue. It is known that bleaching gels diffuse rapidly through the enamel and dentin, and could cause changes in the structure of dentin and pulp tissues, possibly leading to adverse effects ,6. In thMMPs are Ca/Zn-dependent endopeptidases implicated in physiological tissue remodeling, extracellular matrix degradation, tumor growth, and tumor invasion. Moreover, their activity has been attributed to several developmental events involving dental tissues, pathological periodontal processes, dental pulp inflammation, and caries ,23,24,25Based on in-situ zymography results, there were no differences in the dentinal enzymatic activity between the control and experimental groups, regardless of the concentration and pH of the different bleaching agents used. This could be due to the protective effect of the enamel, which was sufficient to avoid the enzymatic activation in the underlying dentin. Recently, Karaarslan et al.  performeCurrent bleaching techniques differ in terms of the type, concentration, exposure time, product presentation, and mode of application ,4,5,6,39The expression of gelatinases MMP-2 and -9 tested in the present study differed with regard to bleaching treatment and exposure time applied. MMP-9, particularly of the pro-form, showed an increased gelatinolytic activity after the application of 10% CP, especially in the protocol with the higher exposure time. Toledano et al.  reportedThe limitation of the present study could be that the human teeth employed in the study were mainly healthy third molars, often extracted operatively, without ever being in occlusal function. Dentin is a very variable tissue that changes during the course of life due to the exposure to the oral conditions, mastication, and microbial attacks ,50. In tThe results of the present study reaffirmed dental bleaching as a safe procedure, also in terms of endogenous dentinal enzymatic activity, if performed according to the recommended protocols, highlighting the importance of protecting exposed dentin tissues before applying bleaching chemicals. However, all the treatments in the present study were performed on healthy unrestored teeth, and further investigations need to be done in order to evaluate the effects of these procedures on teeth restored with direct composite resin fillings.n = 5, sample size calculated using G*Power 3.1.9.7 for Windows with a calculated effect size of f = 1.0269636, α error probability = 0.05, power (1-β error probability) = 0.85) , either stored in water at 4 °C and used within one week, or washed under running water and frozen immediately (−20 °C) and defrosted right before the protocol, were used for the present test. The tooth crowns were separated from the root and cut into four parts  using a slow-speed diamond saw  with water cooling. Three parts from each tooth were used in the study. A blinded researcher (not aware of the testing groups) randomly assigned the tooth slices to 3 different groups . Fifty microliters of the fluorescent gelatin mixture were placed on the top of each polished tooth specimen and was protected with a cover slip. The glass slide assemblies were light-protected and were incubated in a humidified chamber at 37 °C overnight.After the bleaching protocols, the tooth quarters were cut into 1-mm thick specimens, glued to glass slides, and polished up to a thickness of approximately 50 µm. In-situ zymography was performed on the specimens immediately after the treatment, in accordance with Mazzoni et al. ,15. To pThe detection of the endogenous gelatinolytic enzyme activity within the dentin structure was based on the hydrolysis of the quenched fluorescein-conjugated gelatin substrate. The process was evaluated by examining the glass slides with a multi-photon confocal laser scanning microscope , using an excitation wavelength of 495 nm and an emission wavelength of 515 nm.The optical sections (z-stacks of images comprising 15 µm of the sample thickness) were acquired from the different focal planes for each specimen . The fluorescence intensity emitted by the hydrolyzed fluorescein-conjugated gelatin was isolated and quantified using Image J software . The relative amount of gelatinolytic activity was expressed as the integrated density of the pixels of the green fluorescence signal within the dentin structure. The evaluated data were homogenous and normally distributed and, therefore, were statistically analyzed using a one-way analysis of variance (ANOVA) and Bonferroni tests . The significance was preset at α = 0.05.n = 8) were either stored in water at 4 °C and used within one week, or were washed under running water and were frozen immediately (−20 °C) and defrosted right before use. The teeth were ground free of enamel, pulpal soft tissue, and cementum. Dentin powder was obtained by trituration with a Retschmill . Aliquots of 100 mg were prepared in duplicate for each group  was added to the powder and was constantly agitated at 4 °C for 24 h, as well as being protected from the light. The samples were then sonicated in an ultrasound bath with water and ice for 10 min. The vials were centrifuged for 20 min at 4 °C  and the supernatants were collected. The protein content in the supernatants was concentrated using the Vivaspin centrifugal concentrator  for 20 min at 25 °C . The total protein concentration of the dentin extracts was determined by the Bradford assay. Dentin protein aliquots (60 µg) were diluted with a Laemmli sample buffer in a 4:1 ratio. Electrophoresis was performed under non-reducing conditions using 10% sodium dodecyl sulphate-polyacrylamide gel (SDS-PAGE) containing 1 mg/mL fluorescent dye-labelled gelatin. Pre-stained low-range molecular weight SDS-PAGE standards  were used as reference markers. After electrophoresis, the gels were washed for 1 h in 2% Triton X-100 and were incubated in a zymography activation buffer  for 48 h. The proteolytic activity was evaluated and registered with a long-wave ultraviolet light scanner . Gelatinase activities in the specimens were analyzed in duplicate. The densitometric evaluations of the bands obtained from the zymography were performed using the ImageJ software. For all groups, 1.8 mL of the extraction buffer (50 mM Tris-HCl, with a pH of 6.0, containing 5 mM CaClThe bleaching treatments performed on the enamel of sound teeth do not influence the dentinal enzymatic activity. On the other hand, when bleaching agents are applied on unprotected dentin tissue, the MMPs are more expressed, particularly when carbamide peroxide is used, and this effect is related to the exposure time.


Carcinosarcoma of the pancreas is a rare entity with poor prognosis. Here, we report a case of pancreatic carcinosarcoma in a 68-year-old male patient who underwent a pancreatoduodenectomy for a unilocular cystic mass in the head of the pancreas. Histologically, the lesion showed a biphasic tumor with a carcinoma component and a spindle cell sarcomatous component, which were intimately intermingled. Most of the carcinoma components are well-differentiated ductal adenocarcinoma with small areas of moderately to poorly differentiated ductal adenocarcinoma. The sarcomatous component is a high-grade highly cellular spindle cell tumor with frequent mitosis and apoptosis. Immunohistochemical studies demonstrated that the carcinomatous component was positive for epithelial markers and cyclin D1, and the sarcomatous component was negative for these markers while positive for vimentin, p16, and DOG1 with patchy positivity for S100. Other markers, including SOX10, CD117, Melan A, HMB45, actin, desmin, myogenin, beta-catenin, TLE1, and p53, were negative in both components. Molecular studies demonstrated that the tumor was microsatellite stable. Whole exome next generation sequencing analysis was performed and no pathogenic alterations in the genes were identified. Carcinosarcoma of the pancreas is a rare tumor with limited clinical and pathologic data reported in the literature . As per A 68-year-old male with a history of diabetes was admitted to our hospital with a two-week history of abdominal pain, jaundice, nausea, anorexia, and episodes of loose stools. Physical examination revealed right-sided abdominal tenderness. Laboratory examination revealed slightly higher bilirubin levels (0.4 mg/dL), but serum amylase and lipase levels, and complete blood count were all within the normal range. Abdominal computed tomography demonstrated a large cystic mass in the head of the pancreas, which measured 8.1 × 7.5 × 7.4 cm, and dilatation of the common bile duct, measuring 22 mm in diameter. There was also dilatation of the pancreatic duct, measuring 5 mm in diameter. The remainder of the pancreas was grossly unremarkable. Fine needle aspiration (FNA) was performed using endoscopic ultrasound (EUS). The EUS FNA fluid test showed a CEA level > 900 ng/mL, and fluid cytology was negative for malignancy or high-grade dysplasia. Endoscopic retrograde cholangiopancreatography (ERCP) was performed with biliary stent placement, which led to the resolution of his jaundice. An extended pylorus-sparing pancreaticoduodenectomy was performed. The operation was uneventful, and the patient was discharged 4 days after surgery.Gross examination: The pancreatic head was entirely replaced by a mass lesion measuring 8.2 × 7.9 × 7.2 cm and was a unilocular cystic lesion containing gray-green turbid fluid with granular material. The cyst structure appeared to communicate with both the main and side duct branches. The cyst lining was gray-green to yellow, trabecular, and glistening to granular with few fibrous strands that arborized through the cystic structure and anchored at opposing sides of the cyst. Using a standard pancreatic cancer sampling protocol, paraffin-embedded sections of formalin-fixed tissue were studied by routine histology at the Indiana University Pathology Laboratory.Microscopic examination: Histologically, the tumor showed two components composed of an epithelial component and a spindle cell component that were intimately intermingled together. The epithelial component had features ranging from well differentiated to moderately and poorly differentiated pancreatic ductal adenocarcinoma. The majority of the epithelial component was well differentiated with simple small to large ductal structures lined by a single layer of columnar to cuboidal cells, which had small and basally located nuclei with smooth and round nuclear contours and open chromatin. They had a moderate amount of eosinophilic cytoplasm without mucinous content . The modImmunohistochemistry: Extensive immunohistochemical studies were performed at the Indiana University Pathology Laboratory due to the mixed features of the lesion . The epiMolecular study: Molecular analysis of the tumor tissue was first performed by Indiana University Molecular Pathology Laboratory and showed that the tumor was microsatellite stable with no mutation in BRAF, KRAS, and NRAS genes. Additionally, the tumor tissue was sent to the Caris Life Science Laboratory  for next generation sequencing analysis of whole exome sequencing (WES). Direct sequence analysis was performed on genomic DNA using Illumina NovaSeq 6000 sequencers. Tumor mutation burden (TMB) was low and genomic loss of heterozygosity (LOH) was also low, with 10% of the tested genomic segments exhibiting LOH. The whole exome sequencing in our case showed no pathogenic alterations in the genes, such as BRAF, ATM, BRCA1, BRCA2, PALB2, SMAD4, NRG1, and NTRK1/2/3. However, the results for AXL1, HDAC1, MED12, NOTCH1, PIK3CB, POLD2, PRKACA, PTPN11, TERT, and XRCC1 were indeterminate because of the low coverage of exons in these genes.The patient was followed up for three months after surgical resection. The last time he had an appointment for discussing the adjuvant chemotherapy. But he was then lost to follow up without receiving adjuvant chemotherapy.Carcinosarcomas are rare malignant neoplasms. They are histologically characterized by two distinct components: an epithelial component and a mesenchymal component. These dual malignancies most frequently arise in the uterus, ,6, but tHistologically, the tumor in our case showed that the carcinoma and sarcomatous components were intimately intermixed. Most of the carcinoma was well-differentiated ductal adenocarcinoma, with well-formed ducts and bland-appearing epithelial cells. Small scattered areas showed moderately differentiated ductal adenocarcinoma with more complex ductal structures. Small areas showed poorly differentiated epithelial components with infiltrating atypical epithelioid cells, forming small sheets, nests, or single cells. The spindle cell component was highly cellular, with frequent mitosis and apoptosis. These findings suggest a progressive evolution/dedifferentiation of a carcinoma from low-grade to moderate- and high-grade carcinoma and then transition/transformation to a high-grade spindle cell sarcomatous tumor, likely through the mechanism of so-called epithelial–mesenchymal transition. There were foci of hemosiderin deposition suggestive of a previous hemorrhage. It is likely that multiple episodes of hemorrhage and/or necrosis resulted in central cystic formation, as observed radiologically and grossly. No areas showed papillary structures or cells with mucinous features. Intraductal pancreatic mucinous neoplasm (IPMN) often shows positivity for MUC5 and MUC6 in the gastric and pancreaticobiliary types and positivity for MUC2 and MUC5 in the intestinal type, but these markers were all negative in this tumor. Therefore, there was no evidence to suggest that this tumor developed from an IPMN. This was a male patient, and there was no ovarian-type stroma underlying the epithelium. The tumor was negative for the estrogen receptor and inhibin. Hence, there was no evidence to suggest that this tumor was derived from a mucinous cystic neoplasm.Immunohistochemistry studies showed that the adenocarcinoma cells had a positive reaction for epithelial markers, as expected, while no staining of these markers was observed in the sarcomatous component. Carcinosarcoma can be differentiated from sarcomatoid carcinoma which usually shows focal cytokeratin reactivity in the spindle cell component. The spindle cell component of this tumor was positive for DOG1 but negative for CD117. DOG1 (Discovered on GIST-1) is not a specific marker for gastrointestinal stromal tumors (GIST), for it can also be positive in carcinomas of salivary or sweat glands and in sarcomas such as malignant peripheral nerve tumors, desmoplastic/spindle cell melanoma, and prostatic stromal sarcoma ,11. EvenIn our case, the epithelial component was positive for cyclin D1 and negative for p16, while the sarcomatous component was the opposite. The P53 stain pattern was the wild type, with no loss of expression or overexpression in both components. It is known that cell cycle-regulating molecules are essential for the control of cellular proliferation. Two pathways are involved in cell cycle modulation: the p16-cyclin D1/CDK4-pRb pathway and the p53 pathway . AmplifiFor pancreatic carcinosarcoma, available data are limited regarding its treatment and prognosis ,2. The lIn summary, this case highlights the morphology, immunohistochemical characteristics, and molecular study results of pancreatic carcinosarcoma, a rare entity of malignant pancreatic neoplasm. The presence of well to moderately and poorly differentiated ductal adenocarcinoma in close contiguity with a high-grade spindle cell sarcomatous component implies the progressive evolution of the tumor. Molecular studies showed that the tumor was microsatellite stable, and no pathogenic mutation was identified in genes by whole exome sequencing. More studies on this rare but ominous tumor are needed to understand its pathogenesis, treatment, and prognosis.


Thus, soaking is not required for the ETHOS X extraction of polyphenols from LSTT leaves. RSM was successfully applied to explore the impact of ETHOS X extraction conditions and optimize the extraction conditions. Radiation time significantly affects the recovery yield of phenolics (p < 0.05) positively, whereas irradiation power and sample to solvent ratio adversely influenced the extraction yields of phenolics. The optimal ETHOS X extraction conditions were: radiation time of 60 min, irradiation power of 600 W, and sample to solvent ratio of 2 g/100 mL. Under these conditions, 119.21 ± 7.09 mg of phenolic, 85.31 ± 4.55 mg of flavonoids, and 137.51 ± 12.52 mg of proanthocyanidins can be extracted from a gram of dried LSTT leaves. In comparison with UAE and SWB, ETHOS X is not more effective for the extraction of phenolics than UAE and SWB. However, this technique can save half of the solvent volume compared to UAE and SWB techniques.The lemon-scented tea tree (LSTT) is an Australian native herb and is a rich source of essential oil and phenolics. The ETHOS X extraction system is known as a commercial microwave-assisted extraction (MAE) system for extracting bioactive compounds from plant materials. This study investigated the influence of soaking time, radiation time, microwave power, and sample to solvent ratio on the extraction efficiency of polyphenols and antioxidant properties from lemon-scented tea tree leaves and optimized the extraction conditions using response surface methodology (RSM). The effectiveness of ETHOS X was further compared with ultrasound-assisted extraction (UAE) and shaking water bath (SWB) techniques. The results revealed that soaking time did not significantly affect the recovery of phenolics from the leaves ( Phytochemicals derived from plant materials have been used as the key ingredient in many foods, nutraceuticals, and pharmaceuticals products . Of thosMAE is one of the most common advanced techniques applied for the extraction of plant phytochemicals ,9. In thThe LSTT is well known for its strong lemon flavor. It has been used as traditional medicine and in tea blends and is also applied as a natural preservative in food products and as a substitute for lemon flavor in dairy products ,17,18. TThe leaves were arbitrarily collected from the trees on the central coast, Ourimbah, NSW, Australia  in February 2020. After collection, the leaves were immediately transferred to the laboratory, and soaked into liquid nitrogen. The frozen leaves were freeze dried for 48 h using a freeze dryer . The moisture content of the dried ground leaves was 3.2%. A commercial blender  was used to grind the dried leaves. The ground leaves were sieved through a steel standard sieve mesh of 1.4 mm. The fine samples collected after sieving were kept in a labelled, airtight packet at −20 °C for extraction.All of the solvents, chemicals, and reagents used in this study were analytical grade. The organic solvents , vanillin, and sodium hydroxide were obtained from Merck . Folin-Ciocalteu’s reagent and other chemicals including anhydrous sodium carbonate, sodium nitrite, hydrochloric acid, potassium persulfate, copper (II) chloride, ferric chloride, aluminum chloride hexahydrate, ammonium acetate, 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), (±)-6-hydroxy-2,5,7,8-tetramethylchroman-2- carboxylic acid(Trolox), 2,4,6-Tri (2-pyridyl)-s-triazine, neocuproine, gallic acid, and catechin were purchased from Sigma-Aldrich Pty Ltd. v/v) was used as the extraction solvent since a previous study found that it was the most effective solvent for extracting polyphenols and antioxidant potentials from lemon-scented tea tree leaves [The experimental design for this study is shown in e leaves . Two gra1: 40–60 min), microwave power level (X2: 600–1000 W), and sample to solvent ratio (X3: 2–6 g/mL) were assessed using RSM. The predicted yield of phenolics and antioxidants in response to extraction parameter level can be represented by this polynomial functional equation (Equation (1)):The Box–Behnken three-factor, a three-level design involving fifteen experimental runs, was employed  includinY is the predicted response for phenolics and antioxidant capacities, β0 is coefficient for intercept, iβ, iiβ, and ijβ represent the regression coefficients of the linear, quadratic, and interaction effects, respectively; n is the number of variables; and iX and jX are the independent variables [Here, ariables . After oFinally, the optimized MAE conditions were used to prepare a dry phenolic extract for comparison with UAE and SWB. The liquid extracts from MAE, UAE, and SWB were concentrated using a rotavapor  and followed by freeze dying . The dry extracts were diluted using 50% aqueous acetone for the analysis of polyphenols and antioxidant potentials, and the methods were compared based on the level of phenolics and antioxidant potentials of the dry extracts.The UAE and SWB extraction were conducted using conditions described by Saifullah et al. . After eThe quantitative assays of different phenolic groups in the LSTT leaves extract were performed using the UV (Ultraviolet–visible) spectrophotometric methods ) as described in previously reported protocols. Total phenolic content (TPC) analysis was performed as described by AOCS . The TPCAntioxidant properties of the extract were measured by FRAP, CUPRAC, DPPH, and ABTS antioxidant assays. FRAP, DPPH, and ABTS were assayed by following the method described by Benzie and Strain , Brand-Wp-value less than 0.05 (p < 0.05) was considered statistically significant. All of the experiments/data acquisition were conducted in triplicates and the results are expressed as mean ± standard deviation.To observe the effect of soaking time, all pair mean comparison Tukey-Kramer HSD post hoc test was carried out using JMP software . RSM with Box-bhenken design was applied for the experimental design, analysis of variance, and interaction effect analysis, and prediction of optimal conditions using the JMP software. The JMP software was also used to compare the predicted values with the experimental values using a paired comparison analysis. In all assays, the Soaking is known as one of the common conventional extraction techniques . It is ap > 0.05). However, soaking time significantly influenced the antioxidant properties in the extract. The FRAP and CUPRAC were highest at the soaking time of 90 min. The antioxidant capacities were increased 8–13% for 90 min soaking compared to the control sample. However, soaking time doesnot affected the ABTS and DPPH properties significant. The longer soaking time could help to release additional antioxidants to the extract. However, the longer soaking time did not help extract more phenolics from the LSTT leaves. Therefore, soaking is not needed if phenolics are the target compounds for the extraction of LSTT leaves to save time. The following extraction to test MAE impact, therefore, was conducted without prior soaking of the sample.In general, the effects of soaking time on the extraction of major phenolic groups  was insignificant (p < 0.05) influenced the extraction yield of polyphenols and antioxidant properties.The individual, interaction, and quadratic terms effects of MAE parameters on the extraction yield of phenolics and antioxidants are presented in The extraction time in MAE represents the time length the sample is exposed to microwave irradiation. In a long extraction time, the sample is exposed to microwave irradiation longer which causes more ruptures in sample tissues and plant cell walls leading to an increase in phenolics released . Hence, p < 0.05) influence the extraction yield of all tested phenolic groups and antioxidant capacity measured. This indicates that increasing the value of these two parameters decreases the extraction yield of polyphenols and antioxidant properties in the extract. The microwave generates temperature in polar solvents as a result of dipole rotation [From the results, it was found that microwave power and sample to solvent ratio negatively and significantly  can be similarly or oppositely influenced by interaction or combined effects of extraction parameters compared to the effect of a single factor. The interaction effects of time × power and time × sample to solvent ratio significantly affected the TFC yield, and their influence was negative and positive, respectively. On antioxidant capacity, only CUPRAC was negatively and significantly influenced by effects time × power. Among the quadratic effect, only sample to solvent ratio positively and significantly influence the FRAP and DPPH properties of the extract. R2 value, less difference between R2 and adjusted R2, lower RMSE value, insignificant lack of fit, and significant p-value indicates the models are well fitted with experimental data and can be reliable to predict the values of dependent variables [R2 values were ranging from 0.92–0.95, lack of fit values was insignificant (p > 0.05), and the p-value of the model was significant (p < 0.05), which indicates that the polynomial prediction models were reliable to predict polyphenols and antioxidant properties level in MAE derived extract. The lower F ratio and RMSE values also further confirm their validity. The polynomial equations for TPC, TFC, Pro.A, FRAP, CUPRAC, DPPH, and ABTSare Equations (2)–(8).RSM analysis produces a polynomial equation, which represents the relationship between the extraction yields and extraction parameters. ariables ,44,45. It-test. The experimental outcomes should be similar to the predicted yield to consider the optimized conditions are valid. The good correlation between the predicted and experimental outputs confirms the reliability of the model and optimized conditions [p < 0.05) differences between the predicted and experimental values for each assay, suggesting that the predicted optimized MAE parameters values were successfully validated and can be used in practical applications. RSM analysis offers the best optimized MAE parameters values for the maximum yield of targeted phytochemicals and their desired attributes. Based on the prediction of the models, the optimized MAE conditions were radiation time of 60 min, power of 600 W, and the ratio of 2 g/100 mL. To validate these conditions, the obtained MAE parameters value was applied in a practical experiment and the predicted outputs were compared with experimental yields using a statistical analysis paired comparison nditions . The pheThe effectiveness of commercial MAE was compared with UAE and conventional SWB in terms of recovery yield of polyphenols and antioxidant properties in dry extract, and the results are shown in This study revealed that soaking time before MAE has minimal effect on the extraction yield of polyphenols and antioxidant capacities. However, longer extraction/irradiation time has a positive significant influence on the extraction yields. Other extraction parameters, power, and ratio have considerable but negative effects on the phenolics yield. RSM was successfully applied to optimize the extraction parameters, and the validated optimized extraction conditions are time 60 min, power 600 W, and ratio 2 g/mL. RSM analysis indicates the extraction parameters are more significant compared to interaction and quadratic terms. There was no significant difference in extraction efficiency for phenolics and antioxidant capacity among MAE, UAE, and SWB techniques, but MAE requires less solvent for extraction than UAE and SWB. Therefore, these MAE optimal conditions can be applied for the extraction of polyphenols and antioxidant potentials from lemon-scented tea tree leaves. They can be also employed for recovery polyphenols from spent leaves, which are waste and generated from essential oil production. These extracts enriched with polyphenols can be used as functional ingredients for food and pharmaceutical products.