Our results revealed that Guangxi and Guizhou Sui people showed a strong genetic affinity with populations from southern China and Southeast Asia, especially Tai-Kadai- and Hmong-Mien-speaking populations as well as ancient Iron Age Taiwan Hanben, Gongguan individuals supporting the hypothesis that Sui people came from southern China originally. The indigenous Tai-Kadai-related ancestry (represented by Li), Northern East Asian-related ancestry, and Hmong-Mien-related lineage contributed to the formation processes of the Sui people. We identified the genetic substructure within Sui groups: Guizhou Sui people were relatively homogeneous and possessed similar genetic profiles with neighboring Tai-Kadai-related populations, such as Maonan. While Sui people in Yizhou and Huanjiang of Guangxi might receive unique, additional gene flow from Hmong-Mien-speaking populations and Northern East Asians, respectively, after the divergence within other Sui populations. Sui people could be modeled as the admixture of ancient Yellow River Basin farmer-related ancestry (36.2–54.7%) and ancient coastal Southeast Asian-related ancestry (45.3–63.8%). We also identified the potential positive selection signals related to the disease susceptibility in Sui people via integrated haplotype score (iHS) and number of segregating sites by length (nSL) scores. These genomic findings provided new insights into the demographic history of Tai-Kadai-speaking Sui people and their interaction with neighboring populations in Southern China.Sui people, which belong to the Tai-Kadai-speaking family, remain poorly characterized due to a lack of genome-wide data. To infer the fine-scale population genetic structure and putative genetic sources of the Sui people, we genotyped 498,655 genome-wide single-nucleotide polymorphisms (SNPs) using SNP arrays in 68 Sui individuals from seven indigenous populations in Guizhou province and Guangxi Zhuang Autonomous Region in Southwest China and co-analyzed with available East Asians  Southwest China is home to diverse ethnic minorities and linguistic families. Previous population genetic studies based on genetic markers including mitochondrial DNA (mtDNA) and Y-chromosome haplogroups, short tandem repeats (STR), insertion/deletion polymorphisms (InDels), and genome-wide single-nucleotide polymorphisms (SNPs) shed light on the genetic profile and demographic history of ethnolinguistic groups from southern China and Southeast Asia . A SNP c∗, 6.7%; M7b∗, 6.7%; M7b1, 6.7%; and M8a, 6.7%), F , and R ] , as well as in Taiwan aborigines, but not in other East Asians, indicating the Sui was native to southern China . According to the historical accounts, the ancestors of Tai-Kadai-speaking populations were ancient Baiyue tribes, the indigenous people living in southern China. Forced by warfare and famine during the Qin dynasty (circa second century B.C.), Chinese Han continuously expanded toward the south for a long time. A great many Baiyue people migrated to southwest China and then formed the Tai-Kadai-speaking people . Publish, 3.3%)] . From thrn China .Previous genetic findings were predominately based on autosomal/X/Y STRs, mainly aimed to evaluate the forensic characterization of STR markers and investigate the genetic relationships between the Guizhou Sui people and the surrounding Tai-Kadai-speaking, Hmong-Mien-speaking Miao, and Sinitic populations living in southern China . Thus, tA total of 68 blood samples were collected from the seven populations from the Guangxi Zhuang Autonomous Region and Guizhou province with written informed consent and genotyped using Affymetrix WeGene V1 array. These samples were collected randomly from unrelated participants whose parents and grandparents are indigenous people and have a non-consanguineous marriage of the same ethnical group for at least three generations. The ethnicities of all participates were used as their self-declaration based on their family migration history and corresponding family records. Our study and sample collection were reviewed and approved by the Medical Ethics Committee of Youjiang Medical University for Nationalities and Xiamen University  and followed the recommendations provided by the revised Helsinki Declaration of 2000. After removing batch effects and missing sites, we genotyped 498,655 genome-wide SNPs. We listed the detailed sample information in via the GCTA software . Two datasets were used in subsequent population genetic analysis: (1) we merged our newly collected Sui individuals with a 1240K capture dataset to create the high-SNP-density “merged-1240K” dataset harboring 373,933 SNP sites; and (2) we merged our newly published Sui people data with 82 present-day populations or nine meta-populations and 40 ancient groups from the Affymetrix Human Origins (HO) panel to generate the “merged-HO” dataset, covering lower SNP sites  but maximum the number of populations and individuals. Data merging was done by mergeit from EIGENSOFT  calculated the shared genetic drift between X and Y since their divergence from the outgroup. Admixture-f3 evaluated the admixture signals in the targets (Z-score < −3). In the form of f4, a Z-score > 3 implied that W shared more alleles with Y than with X; a Z-score less than −3 suggested that W shared extra alleles with X compared with Y; a | Z-score| < 3 indicated that X and Y formed a clade in relation to the outgroup and W. We used an African population Yoruba as the outgroup.We used the MIXTOOLS  with defqpWave as implemented in ADMIXTOOLS  were accepted.We used MIXTOOLS  with defWe used ancient Northern East Asian-related ancestry (represented by YR_LN) and ancient Southeast Asian-related ancestry (represented by Liangdao2) as sources. Seven populations  were used as outgroups.We further used YR_LN, Ami, Vietnam_N as proxies for ancient Northern East Asian, ancient coastal Southeast Asian, and ancient inland Southeast Asian-related ancestries. Nine populations  were used as outgroups.via the selscan software with default parameters  ia Kobas .We first carried out PCA to uncover an overview of the genetic structure of East Asians and Southeast Asians . The topThe distributions of the studied Sui people were relatively scattered, with two major clusters: one genetic cline consisting of Huanjiang Sui individuals from Guangxi, which was placed on the Hmong-Mien-related cline; and the other comprising the rest of the newly studied Sui groups, clustered together with published Tai-Kadai-speaking populations and displaying closer genetic relationships with AA, AN, and Sinitic-speaking populations from Southern China and Southeast Asia. Yizhou Sui from Guangxi slightly shifted toward the Han-related cline. Furthermore, we detected that the Sui-related cluster tended to deviate to HM-related cline compared with other published TK speakers; Tibetan-Burman-related and Altaic-related groups exhibited significant genetic differentiation with the studied Sui people. After projecting the ancient EA and SEA individuals onto the top two PCs, we observed that most ancient individuals fall relatively close with geographically close modern populations. Coastal Fujian_EN , Fujian_LN , Taiwan_IA (Taiwan_Hanben and Taiwan_Gongguan), Inland Vietnam Bronze Age (Vietnam_BA_DongSonCulture) individuals plotted relatively close with our studied Sui populations than other published ancient samples.K = 4 revealed the difference in genetic compositions within Sui populations. The studied Huanjiang Sui individuals were characterized by a considerable amount of Hmong-Mien-related ancestry (∼70%), with ∼22% Inland Southeast Asian-related ancestry, ∼7% coastal Southeast Asian-related ancestry, and very few Northern East Asian-related ancestry (less than 1%). While the primary ancestry component assigned to other studied Sui groups as well as neighboring TK and AA speakers was the Inland southeast Asian-related component, they harbored less Hmong-Mien-related ancestry but higher proportions of coastal Southeast Asian-related and Northern East Asian-related ancestry compared with Huanjiang Sui. In addition, Yizhou Sui harbored a significantly higher Northern East Asian-related component than each Sui population .Model-based ADMIXTURE with an optimal K of 4  suggesteTo explore the genetic affinity between the studied Sui and reference populations, we first constructed the unrooted N-J phylogenetic tree based on Wright’s fixation index pairwise Fst genetic distance among 79 modern populations . We idenf3-statistics in the form of f3 to measure the shared genetic drift. The cluster patterns in the heatmap (f3: 0.237811), followed by Hmong (f3: 0.235770) and then by other Sui people (f3: 0.222949–0.225170). Focusing on the results of outgroup-f3 (f3 > 0.223575), followed by Fujian Historical Chuanyun individuals (f3 > 0.220979), Fujian_LN Xitoucun individuals (f3 > 0.220718), and inland YR basin farmer populations (f3 > 0.215268).We subsequently conducted outgroup- heatmap  confirme Yoruba) , we founf4-statistics in the form of f4 shown in f4 values with absolute Z-scores larger than 3 indicated that X shared more genetic drift with Sui population 1 relative to Sui population 2; otherwise, significant positive f4 values indicated more shared alleles between X and Sui population 2 rather than Sui population 1. Z-scores ranging from  denoted that Sui 1 and Sui 2 formed one clade in relation to X and outgroup Yoruba, respectively. The observed significant Z-scores in f4 suggested that HM-speaking Hmong, Miao_Huanjiang, and PaThen shared excess alleles with Huanjiang Sui individuals (−18.139 ≤ Z-scores ≤ −4.691). Tai-Kadai-speaking Li, Mulam, Nung, and Tay and AA-speaking Kinh and Muong shared fewer alleles with Huanjiang Sui than with other Sui people (1.138 ≤ Z-scores ≤ 3.738). The significant negative Z-scores in f4 indicated that there are significantly more derived alleles shared between Northern East Asians  and Guangxi Yizhou Sui compared with Sui_Libo , Sui_Dushan (Z-scores = −3.085), and Sui_Duyun (Z-scores = −3.000). AN-speaking Ede shared excess alleles with Sui_Duyun (Z-scores = 3.101) and Sui Libo (Z-scores = 3.048) compared with Sui Yizhou. HM-speaking Hmong shared more derived alleles with Sui_Duyun than with Sui_Yizhou (Z-scores = 3.355). We did not identify the genetic difference among Guizhou Sui groups and Guangxi Sui_Sandu as no f4 with significant Z-scores were observed.To quantitatively evaluate the genetic similarity and differentiation among the studied Sui populations compared with the worldwide reference populations, we performed symmetrical qpWave analysis, which was more accurate than symmetry f4-statistics, to further test whether seven Sui groups were genetically homogeneous  in the one-way admixture model when Guangxi Nandan, Huanjiang, and Yizhou Sui were used as one of the test populations. Pairwise qpWave results confirmed that (1) there is genetic homogeneity within Guizhou Sui individuals; (2) there is genetic heterogeneity within Guangxi Sui individuals; and (3) Guizhou Sui and Guangxi Sui were not derived from a single homogenous population in relation to the outgroups we used.We then performed pairwise ogeneous . Each paqpWave test  in the “outgroup-dropping pairwise qpWave test” instead, suggesting that the “dropped outgroup”-related ancestry might have a unique gene flow with one of the test groups, explaining the nonhomogeneity between the pairwise test populations. The results suggested that different levels of Ami-related gene flow may lead to the heterogeneity between Guangxi Sui and Guizhou Sui; coastal Amur River Neolithic DevilsCave-related ancestry may drive the nonhomogeneity between Yizhou Sui and other Sui groups.We next did the outgroup-dropping pairwise ave test . Pairwisf4-statistics in the form of f4 for each Sui group to explore the possible extra gene flow that Sui people/Y received from X after the divergence between the specific population Y and studied Sui.We performed all possible Z-scores suggested that Hmong-Mien speakers  and Tibetan-Burman-related populations  shared more derived alleles with our newly reported Sui people, indicating that the Sui people received the extra Hmong-Mien-related ancestry and northern East Asian sources after the separation of the Hlai and Sui groups or explaining that the Hlai received the ancestry which was a deeper linage than the common ancestor of Hmong-Mien-speaking populations and Sui people. No significant positive f4 values were observed.When Tai-Kadai-speaking Hlai (representing the unadmixed form of Tai-Kadai-speaking populations) was same as Y , the obsoutgroup-f3 statistics, the geographically close Tai-Kadai-speaking Maonan firstly clustered with Sui groups. Thus, we further did the formal test in the form of f4 to explore the fine-scale genetic differentiation between the studied Sui and Maonan (Z-scores| < 3). Hmong-Mien-speaking Miao groups from Vietnam and Guangxi showed additional gene flow with Guangxi Sui_Huanjiang (−17.884 ≤ Z-scores ≤ −17.838). Hmong shared more alleles with Sui_Duyun than with Maonan (Z-score = −3.044). Some of the Tai-Kadai speakers , Austroasiatic speakers (such as Jing), and Austronesian speakers (such as Ede) shared more alleles with Maonan than with the Sui_Huanjiang/Sui_Yizhou/Sui_Duyun (Z-scores ≥ 3.027). When X = ancient EA and SEA individuals, we observed that ancient Fujian_EN (Liangdao2) individuals shared more genetic drift with Maonan than with each newly studied Guangxi Sui .As shown by the Fst-based N-J tree and via admixture-f3-statistics. The statistically significant negative f3 values with Z-scores less than −3 suggested that the target population might be an admixture of two source-related populations. Here, we reported the top 10 lowest f3 value results for each Sui group in the form of f3 and f3, respectively, in We exhausted all possible pairs of reference populations as genetic sources to estimate admixture signals in each studied Sui population f3, only three significant admixture signals were observed in Sui_Duyun when we used Hmong-Mien ancestry (Hmong) as one source and TK-related (Li/Mulam) or AA-related ancestry (Muong) as the other source (Z-scores ≤ −3.093). Although no more significant negative f3 values were identified when we used other Sui groups as targets, it is important to note that a nonnegative admixture-f3 value does not prove that there is no admixture. The lowest admixture-f3 values were achieved in each studied Sui group except Yizhou Sui when we used the same pairs of source populations as follows: (1) Hmong-Mien-related Miao_Huanjiang/Hmong as source1 and Tai-Kadai related Li/Mulam/Gelao as source2; and (2) Hmong-Mien-related Miao_Huanjiang/Hmong as source1 and AN related Ami/Atayal as source2. For each newly studied Guizhou Sui group , when we used Hmong-Mien-related Miao_Huanjiang/Hmong as source1 and AA-related Muong as source 2, low f3 values can be produced. For Sui_Yizhou, when one of the source populations represented Sino-Tibetan-related ancestry (Sherpa/Tibetan_Shigatse) and the other represented TK-related (Li) or AN-related ancestry , top negative f3 values were generated.When focused on f3, the negative f3 values were observed in all Sui groups when one source was from ancient Southeast Asia  and the other from ancient Northern East Asia (such as Miaozigou_MN and AR_IA), suggesting the north–south admixture pattern for Sui people.When focused on the form qpAdm to explore the plausible admixture models for our studied Sui populations. We used the Late Neolithic Yellow River Basin farmer-related population (YR_LN) and Early Neolithic Coastal Liangdao2 individuals as proxies for the Northern East Asian-related and Southeast Asian-related source populations in a two-way admixture model , and Sui_Nandan  samples harbored a similar proportion of Liangdao2-related ancestry. The Yizhou Sui had the highest proportion of YR_LN-related ancestry  among Sui groups.We further applied re model . We obseadmixture-f3-statistics suggested, our studied Sui might be modeled as an admixture of one ancient inland Southeast Asian group and one ancient North East Asian group. Therefore, we used coastal Southeast Asian , inland Southeast Asian (represented by Vietnam_N), and Northern East Asian (represented by YR_LN) as three proxies of the possible ancestral sources to infer ancestry mixture coefficients in a three-way admixture model. The studied Sui groups covered similar proportions of coastal SEA ancestry and inland SEA ancestry and clustered with neighboring HM-speaking populations and TK-speaking populations in the ternary diagram  the genetic relationships between the Sui and reference East Asians; (2) the fine-scale population structure within the Sui people; (3) the admixture history of each Sui population; and (4) the potential positive selection signals of Sui people. The patterns of shared genetic drift measured These results were in accordance with the genetic findings based on forensic-related genetic markers  indicating that the Sui people displayed a close affinity with geographically close populations, especially Qiandongnan Miao , AA-speaResults of PCA and model-based ADMIXTURE analysis revealed the genetic substructure within seven studied Sui populations: (1) the TK-speaking Sui individuals from Guangxi Huanjiang formed a cline which was localized on the intermediate of Hmong-Mien-related genetic cline, rather than clustered with neighboring TK-speaking populations, and (2) a relatively loose cluster which consisted of the rest of the newly reported Sui populations, partially overlapped with TK-speaking populations from Southern China and Southeast Asia.f4-statistics and pairwise qpWave consistently showed that the Guizhou Sui people were relatively homogeneous and showed similar genetic profiles with Tai-Kadai-related populations, such as Maonan. While Guangxi Sui groups were relatively heterogeneous, we observed excessive genetic affinity between Tai-Kadai-speaking Huanjiang Sui and the geographically close Hmong-Mien-speaking Miao people although the two groups were ethnically different. Huanjiang Sui did not share the most derived alleles with other Guizhou and Guangxi Sui people, which belonged to the same ethnic group, suggesting that Huanjiang Sui received a significantly geographically close Hmong-Mien-speaking Miao-related ancestry (represented by Huanjiang Miao) after Huanjiang Sui and other Sui people separated from the common ancestor. Sino-Tibetan-related populations contributed to the extra ancestry to Yizhou Sui people compared with other Sui groups, indicating that the Yizhou Sui have been primarily affected genetically by the surrounding Han populations. These results suggested the differentiated demographic history among the studied Sui populations.Symmetric Z-scores of f4 revealed that the ancestors of the Sui people might experience excessive admixture events with HM and NAEs after the divergence with the unmixed, indigenous TK-speaking proxy Li islanders. The significant negative values in admixture-f3 suggested that the ancestor of HM-related populations might also participate in the formation of the Sui people. Hmong-Mien-speaking populations, such as Miao and Yao, are the dominant ethnic groups in southwest China. While the Sui has a relatively small population, there is only one autonomous country for Sui people in China . Previous studies suggested that Hmong-Mien-related people might be the direct descendants of Daxi-related people as there was a rare Y-chromosome haplogroup O3d detected in Neolithic Yangtze river Daxi culture-related people and modern Hmong-Mien speakers  and ancient southeast Asians (ASEAs), in which ANEAs were represented by Neolithic Yellow River Basin-related farmer populations and ASEAs were characterized by Neolithic/modern Coastal Southern East Asians. More specifically, in the three-way admixture model, Sui people and neighboring HM- and TK-speaking populations derived their ancestry from more similar (but still different) proportions of ANEA-, coastal SEA-, and inland SEA-related components compared with the Han from Southern China. Conclusively, the formation of the population structure of Tai-Kadai-speaking populations might be plausibly explained by (1) the differential proportions of the primary ancient sources and (2) the various levels of gene flow with surrounding people, such as HM-speaking and Sinitic-speaking populations.The significant negative speakers . A possivia normalized iHS and nSL. Notably, lots of significant SNPs enriched in the KEGG pathway associated with the susceptibility of complex diseases (such as the Type 1 diabetes mellitus pathway and gastric cancer pathway). When focused on the SNPs which were famous for the natural selection in East Asians in previous genetic studies , rs4148211 (chr2:44071742) on the ABCG8 gene (associated with lipid metabolism), and rs1229984 (chr4:100239318) located in the ADH1B gene , which were likely under natural selection in Sui people .Finally, we detected the potential positive selection signals based on the phased data of Sui people  studies , we obsevia next-generation sequencing (NGS) and single-molecule real-time sequencing (SMRTS) of Sui individuals from more geographic locations  are expected to be conducted to provide genomic insight into the formation of Sui people and to dissect the complex demographic history of populations from Southern China and Southeast Asia comprehensively.However, more population genetic studies based on the whole-genome sequence (WGS) data https://zenodo.org/record/5483577, doi: 10.5281/zenodo.5483577.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: The studies involving human participants were reviewed and approved by Medical Ethics Committee of Youjiang Medical University for Nationalities and Xiamen University . The patients/participants provided their written informed consent to participate in this study.C-CW and XH designed the study. RuW and C-CW wrote the manuscript. XB, YH, RoW, and XH collected the samples. KZ, XY, HM, GH, JG, JZ, MY, JC, XZ, LT, and YL conducted the experiment. RuW and C-CW analyzed the data. All authors reviewed the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer SW declared a past co-authorship with the authors XY, GH, JG, JZ, and C-CW to the handling editor.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Specific metabolic phenotypes of breast cancer result from local interactions such as cancer-adipocyte cross-talk and systemic metabolic influences such as obesity. Here we examined key regulatory enzymes involved in glucose metabolism in breast cancer tissue and cancer-associated adipose tissue of normal-weight and overweight/obese premenopausal women in comparison to benign breast tumor tissue and adipose tissue of weight-matched women. We show a simultaneous increase in 5′-AMP-activated protein kinase (AMPK) protein expression with glucose utilization favoring glycolysis and pentose phosphate pathway in breast cancer tissue. In parallel, we show an increased AMPK protein expression with glucose utilization favoring the pentose phosphate pathway in cancer-associated adipose tissue. Moreover, specific features of cancer tissue glycolysis and glycogen metabolism differ between normal-weight and overweight/obese women. The results suggest context-dependent induction of tissue-specific Warburg effect in breast cancer and cancer-associated adipose tissue.Typical features of the breast malignant phenotype rely on metabolic reprogramming of cancer cells and their interaction with surrounding adipocytes. Obesity is strongly associated with breast cancer mortality, yet the effects of obesity on metabolic reprogramming of cancer and cancer-associated adipose tissue remain largely unknown. Paired biopsies of breast tumor tissue and adipose tissue from premenopausal women were divided according to pathohistological analyses and body mass index on normal-weight and overweight/obese with benign or malignant tumors. We investigated the protein expression of key regulatory enzymes of glycolysis, pentose phosphate pathway (PPP), and glycogen synthesis. Breast cancer tissue showed a simultaneous increase in 5′-AMP-activated protein kinase (AMPK) protein expression with typical features of the Warburg effect, including hexokinase 2 (HK 2) overexpression and its association with mitochondrial voltage-dependent anion-selective channel protein 1, associated with an overexpression of rate-limiting enzymes of glycolysis (phosphofructokinase 1—PFK-1) and pentose phosphate pathway (glucose-6-phosphate dehydrogenase—G6PDH). In parallel, cancer-associated adipose tissue showed increased AMPK protein expression with overexpression of HK 2 and G6PDH in line with increased PPP activity. Moreover, important obesity-associated differences in glucose metabolism were observed in breast cancer tissue showing prominent glycogen deposition and higher glycogen synthase kinase-3 protein expression in normal-weight women and higher PFK-1 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein expression in overweight/obese women. In conclusion, metabolic reprogramming of glycolysis contributes to tissue-specific Warburg effect in breast cancer and cancer-associated adipose tissue. Breast cancer incidence has been rising in recent decades , making Recent studies show that glycolytic reprogramming serves multiple purposes in cancer, including energy production, supply of metabolic intermediates for biosynthetic pathways, posttranslational regulation through glycoprotein synthesis, and redox homeostasis maintenance . AlteredMetabolic reprogramming is no longer viewed as an exclusively intrinsic trait of cancer cells. Rather, metabolic reprogramming is a context-dependent and dynamic process resulting from bidirectional interactions between cancer cells and their local and systemic environment . The tumOur previous studies indicated strong lactate-centric cooperation between breast cancer and cancer-associated adipose tissue in premenopausal women. Our data showed that adipose tissue-expressing lactate dehydrogenase B was found in close proximity to cancer tissue rich in lactate and expressing lactate dehydrogenase A . MoreoveOur current study aimed to uncover the relationship between key features of glucose metabolism in breast cancer and cancer-associated adipose tissues. We investigated the context-dependent nature of glucose metabolism by undertaking a cross-examination of breast cancer and adipose tissue biopsies from normal-weight and overweight/obese premenopausal women. We analyzed protein expression of key rate-limiting and regulatory enzymes involved in major pathways of glucose metabolism, including glycolysis, PPP, and glycogen metabolism, as well as hallmarks of the Warburg effect such as HK 2 protein expression and cellular localization. Our results reveal parallel increases in 5′-AMP-activated protein kinase (AMPK) protein expression levels in breast cancer and cancer-associated adipose tissue, as well as the induction of tissue-specific Warburg effects. In addition, the results strongly support the concept that obesity can directly influence key features of glucose metabolism in breast cancer tissue of premenopausal women and add to our understanding of metabolic reprogramming in the breast cancer microenvironment.2) or overweight/obese (BMI ≥ 25 kg/m2). All women were scheduled for surgical removal of benign or malignant breast tumors. Under general balanced anesthesia, two tissue samples were taken from each patient; one of the breast tumor tissue and one of the breast adipose tissue. Breast (mammary) adipose tissue that was proximal to the tumor but did not include the invasive front of the tumor was designated as tumor-associated adipose tissue. Following the histopathological analysis, samples were classified into either benign breast fibroadenomas or malignant breast invasive ductal carcinomas , thus forming four groups of samples (n = 9): benign tumors from normal-weight individuals, benign tumors from overweight/obese individuals, malignant tumors from normal-weight individuals, and malignant tumors from overweight/obese individuals. Each sample of tumor and adipose tissue was cut into two parts for protein expression/localization analyses, either quantitative (western blotting) or cell type/localization (immunohistochemistry/immunofluorescence). For western blotting, one piece of the tissue sample was immediately frozen in liquid nitrogen. Samples were stored at −80 °C until subsequent protein isolation . For immunohistochemical and immunofluorescent analyses, the second piece of the tissue sample was immediately fixed in 4% paraformaldehyde (PFA) solution and stored in ethanol at 4 °C before downstream histological processing.Thirty-six premenopausal women with regular menstrual cycles for at least six months were included in the study. According to the World Health Organization criteria , particiWestern blot analysis was done for the determination of protein content according to the previously published protocol . PrimaryPeriodic Acid-Schiff (PAS) staining for the histological detection of glycogen was performed according to the manufacturer’s instructions . To detect glycogen-specific staining, pretreatment with a diastase kit containing α amylase was used according to the manufacturer’s instructions . For each sample, pairs of serial sections were used for PAS staining, where one section was pretreated with diastase and served as a negative control for its respective serial section. Five µm thick paraffin-embedded serial sections were used for the staining.Immunohistochemistry was used to confirm cell-specific protein expression and localization of HK 2 in cancer cells and adipocytes in breast tumor and adipose tissue. 4% PFA-fixed samples were embedded into the paraffin and cut into 5µm tick serial sections. The immunolabeling procedure was performed following a previously published protocol  using pr−1; ab46545, Abcam), HK 1 , and HK 2  and the appropriate fluorochrome-conjugated secondary antibodies Alexa Fluor 488 and Alexa Fluor 633 . Sytox Orange  was used as a counterstain to visualize the nuclei. Mowiol  was used as a mounting agent. Leica TCS SP5 confocal laser scanning microscope  was used in sequential mode to acquire confocal images and avoid bleed-through between different channels. The same procedure with the omission of primary antibodies was used as a negative control to test the specificity of antibodies.Immunofluorescence and confocal microscopy were used to analyze the cellular localization and colocalization patterns of relevant proteins following a previously published procedure. Briefly, a sequential immunofluorescence assay was performed in the dark on PFA-fixed, paraffin-embedded 5µm tick serial sections  using prp < 0.05.All data were statistically analyzed using GraphPad Prism software . All data sets were tested for the normality of distribution using D’Agostino or Pearson’s omnibus normality test. To compare within-group variation, a one-way two-tailed analysis of variance (ANOVA) was applied. After assessing the overall differences by the F test, the comparisons of inter-group differences were evaluated by multiple comparison Tukey’s post hoc test. Statistical significances were considered at Assessment of the protein expression levels of the main energy sensor regulating cellular energy metabolism, AMPK as well as the rate-limiting enzymes within three major pathways of glucose metabolism  revealed profound changes in the energy metabolism of malignant tumor tissue in comparison to benign tumor tissue. Consistent with the high energy demands of cancer tissue, AMPK protein expression was significantly higher in malignant tumor tissue than in benign tumor tissue, irrespective of obesity A. SimilaTo investigate the metabolic features of cancer-associated adipose tissue from normal-weight and overweight/obese women, protein expression analysis of key metabolic enzymes in glucose metabolism was performed using western blotting. In comparison to the breast adipose tissue of women with benign tumors, malignant tumor-associated adipose tissue showed higher protein expression levels of AMPK and adipocyte-specific isoform of hexokinase—HK 2 A,B, whilTo assess the glycogen content and analyze glycogen localization in tumor tissues, we performed periodic Acid-Schiff (PAS) staining. Glycogen deposition was observed in benign tumor tissues of both normal-weight and overweight/obese women and was primarily localized in breast epithelial cells . InteresWe performed immunohistochemistry on tissue sections of benign and malignant breast tumor samples from normal-weight and overweight/obese individuals to assess any changes in HK 2 expression in tumor tissues and confirm adipocyte cell-specific localization of HK 2 in tumor-associated adipose tissue. As expected, breast cancer cells showed strong immunopositivity for HK 2 compared to benign tissues in both normal-weight and overweight/obese individuals . MoreoveTo analyze if metabolic reprogramming involving overexpression of HK 2 in malignant tissues is associated with its functional translocation to mitochondria, we analyzed colocalization patterns of HK 1 and HK 2 in relation to VDAC, a mitochondrial marker by immunofluorescent staining and confocal microscopy . The immThis study evaluated hallmark signatures of glucose metabolism in breast cancer and cancer-associated adipose tissue of normal-weight and overweight/obese premenopausal women. Regardless of obesity, increased AMPK protein expression was found in both breast cancer tissue and cancer-associated adipose tissue, synchronous with the induction of glucose utilization. Indeed, breast cancer tissue showed HK 2 overexpression and its association with a mitochondrial marker VDAC followed by increased PFK-1 and G6PDH protein expression. These changes indicate the induction of specific Warburg effect favoring glycolysis and pentose phosphate pathway, respectively. In parallel, cancer-associated adipose tissue showed overexpression of HK 2 and G6PDH, indicating metabolic reprogramming favoring the pentose phosphate pathway. Interestingly, some aspects of glucose metabolism differed depending on obesity . Breast cancer tissue showed abundant cytoplasmic glycogen deposition and higher GSK-3 protein expression in normal-weight women. In comparison, breast cancer tissue showed no prominent glycogen deposition but displayed higher PFK-1 and GAPDH protein expression in overweight/obese women. These results suggest context-dependent metabolic reprogramming in breast cancer tissue and the induction of tissue-specific Warburg effect in cancer and associated adipose tissue.AMPK is a metabolic sensor and a master regulator of energy homeostasis whose activation stimulates catabolism acting on glucose uptake, glycolysis, oxygen consumption, fatty acid oxidation, lipolysis, and mitochondrial biogenesis ,32,33,34The Warburg effect refers to specific metabolic reprogramming where cancer cells rely on increased glucose uptake and glycolytic flux with lactate production even in the presence of oxygen to support growth and proliferation . OverexpOur results show that some aspects of glucose metabolism in breast cancer tissues are specific to the obesity of premenopausal women. Namely, cross-examination of breast cancer tissues from normal-weight and overweight/obese premenopausal women revealed differences in protein markers of glycolysis and glycogen metabolism. Breast cancer tissues of normal-weight women showed abundant cytoplasmic glycogen deposition and increased GSK-3 protein expression compared to overweight/obese women, possibly indicating important obesity-related differences in glycogen metabolism. Previous studies showed cytoplasmic glycogen accumulation in breast cancer . AdditioThis study evaluated protein expression of AMPK, key rate-limiting and regulatory enzymes of glycolysis, pentose phosphate pathway, and glycogen metabolism in breast cancer and cancer-associated adipose tissue in normal-weight and overweight/obese premenopausal women. We show the simultaneous induction of AMPK protein expression and tissue-specific Warburg effects in breast cancer and associated adipose tissue, opening the question of novel roles for AMPK in glycolytic reprogramming in cancer. Namely, increased glucose utilization favors glycolysis and pentose phosphate pathway in breast cancer tissue, with typical features of the Warburg effects including overexpression and mitochondrial localization of HK 2. In contrast, increased glucose utilization in adipose tissue mainly favors the pentose phosphate pathway. Moreover, certain aspects of glucose metabolism seem to be obesity-dependent. Breast cancer tissue in normal-weight women shows more pronounced glycogen deposition, while breast cancer tissue in overweight/obese shows higher protein expression of PFK-1 and GAPDH glycolytic enzymes. Parallel reprogramming of cancer cells and cancer-associated adipose tissue down specific metabolic phenotypes may reveal new aspects of metabolic coupling in breast cancer. Moreover, important obesity-related changes to the glucose metabolism of breast cancer tissue could inform the discovery of novel targets for personalized diagnostic and therapeutic approaches. Our future studies will be directed towards revealing the mechanism of possible AMPK involvement in glycolytic reprogramming as well as fatty acid metabolism and oxidative phosphorylation in breast cancer and cancer-associated adipose tissue.


Human respiratoryviral infections are the leading cause of morbidityand mortality around the world. Among the various respiratory viruses,coronaviruses  have created the greatest challengeand most frightening health threat worldwide. Human coronavirusestypically infect the upper respiratory tract, causing illnesses thatrange from common cold-like symptoms to severe acute respiratory infections.Several promising vaccine formulations have become available sincethe beginning of 2021. Nevertheless, achievement of herd immunityis still far from being realized. Social distancing remains the onlyeffective measure against SARS-CoV-2 infection. Nanobiotechnologyenables the design of nanobiosensors. These nanomedical diagnosticdevices have opened new vistas for early detection of viral infections.The present review outlines recent research on the effectiveness ofnanoplatforms as diagnostic and antiviral tools against coronaviruses.The biological properties of coronavirus and infected host organsare discussed. The challenges and limitations encountered in combatingSARS-CoV-2 are highlighted. Potential nanodevices such as nanosensors,nanobased vaccines, and smart nanomedicines are subsequently presentedfor combating current and future mutated versions of coronaviruses. However, with morethan 2.5 million deaths to date, there is an urgent need for fastand reliable diagnostic and therapeutic approaches against SARS-CoV-2infections. Many aspects of the currently available vaccine formulationsremain to be clarified, including the safety of administration onthe pediatric population and their effectiveness against emergingviral strains.Coronavirus pandemicshave emerged rapidly in the 21st century,with catastrophic consequences.9 Nanomaterials have been developed to combat viral, bacterial, andfungal infections10 because of their uniquephysicochemical characteristics, such as high surface area, nanoscaledimensions, and readily achievable surface modifications. These propertiesenable scientists to improve drug pharmacokinetics, control drug release,enhance drug solubility, facilitate cellular membrane passage, andenhance the bioavailability of pharmaceutics against a series of virusessuch as human immunodeficiency virus, herpes simplex virus, and hepatitisB virus.12 Nanomaterials are promising tools for thediagnosis and treatment of COVID-19.Nanotechnology has opened up new horizons inmany different aspectsof medical science, such as targeted gene delivery, targeted drugdelivery, biosensor platforms, imaging, and diagnosis.The present review systematicallyoutlines the recent advancesreported in the literature on the use of nanoparticles as effectivediagnostic and antiviral treatment tools against recently mutatedcoronaviruses. In addition, an overview of the biological propertiesof all human coronaviruses is provided, with evaluation of their differencesand site-specific infection of the human body. The challenges andlimitations encountered by this technology are discussed. Nanotechnologyoffers multiple roles in combating coronavirus infections, such asnanosensors, nanobased vaccines, and smart medicine.214 HCoV-229E,15 HCoV-NL63,15 HCoV-OC4),16 HCoV-HKU1,17 MERS-CoV,18 and SARS-CoV-2.19 The HCoV-229E and HCoV-NL63 are identified as Alphacoronaviruses, whereas HCoV-HKU1, HCoV-OC43, SARS-CoV1, SARS-CoV2, and MERS-CoVare classified as Betacoronaviruses. havebeen identifiedthat infect humans. They belong to the family Coronaviridae and includeSARS-CoV-1,aviruses. As a cor23 The CoV genome is about26.4–31.7 kb, which is the largest among RNA viruses with guanineand cytosine contents varying from 32 to 43%.24 Genomic RNA acts as a mRNA (mRNA) which plays a key role in thereplication of the viral genome and production of new infectious virusparticles.25 The 5′ untranslatedregion (5′ UTR) and 3′ untranslated region (3′UTR) are the regions of mRNA involved in many regulatory aspects ofgene expression with a major role in RNA–RNA interactions forbinding with viral and cellular proteins.26 A typical CoV comprises at least six-open reading frames (ORFs).Two-thirds of the genome consists of ORF1a and ORF1b, which producetwo polypeptides, pp1a and pp1ab. These polypeptides are processedby viral proteases , main protease(Mpro) and papain-like protease (PLpro)) for cleaving the 16 nonstructuralproteins (NSPs) that are involved in genome transcription and replication.27 The sizes of the NSPs vary in different CoVstrains.21Coronaviruses are enveloped, positivelysensed, single-strandedRNA with spherical capsids (120–160 nm) that collectively resemblea crown with a solar shape.28 The S proteinis a large glycosylated transmembrane protein (1160–1400 aa)that plays an essential role in the recognition of cellular receptorsfor infection of a susceptible cell. The size of this protein differsamong the coronaviruses: 21493 aa, 1270 aa, and 1273 aa for SARS-CoV-1,MERS-CoV, and SARS-CoV-2, respectively.21 The E protein is a small envelope protein (74–109 aa) responsiblefor the assembly of virions and curving of the viral envelope.29 The M protein is an integral glycoprotein (250aa), which has three transmembrane regions and interacts with otherstructural proteins to maintain the virion structure.30 The N protein is a heavily phosphorylated nucleocapsidprotein (500 aa), which has a key role in encapsulating the viralgenome into helical nucleocapsid within the viral particles.31 The arrangement of N, E, and M proteins amongcoronaviruses is different, as shown in 33 The Betacoronavirus genus has an additional structural protein, hemagglutinin-esterase and a transmembrane protein that forms homodimers.33 The HE protein has acetyl-esterase functionthat is not necessary for in vitro viral replication. However, theHE protein may affect early viral infection in vivo by binding reversiblyto O-acetylated sialic acids. The 3a/b and 4a/b proteins are othermature proteins responsible for various important functions in virusreplication and genome maintenance.27Coronaviruses have fourcanonical structural proteins includingspike protein (S), envelope protein (E), membrane protein (M), andnucleocapsid protein (N). Besides, there are several nonstructuralproteins that are encoded by ORFs 10 and 11 on one-third of the genomenear the 3′ end.34 human aminopeptidase N (CD13) by HCoV-229E,36 dipeptidyl peptidase 4 (DPP4) by MERS-CoV,37 and angiotensin-converting enzyme 2 (ACE2) by HCoV-NL63, SARS-CoV1,and SARS-CoV2.38 In addition, protease can helpCoVs enter cells. For example, transmembrane protease serine 2 (TMPRSS2)and airway trypsin-like protease TMPRSS11D activate the S proteinin HCoV-229E, SARS-CoV-1 and SARS-CoV-2 infections,39 while cathepsin L is activated in SARS-CoV and MERS-CoV.40 After the virus enters a susceptible cell, thegenome is transcribed and translated. Replication and transcriptionof the coronavirus genome occur with continuation/discontinuationof RNA synthesis that is mediated by a huge replicase complex.41 The replicase complex is about 20 kb and containsup to 16 viral subunits along with a number of host cellular proteins.42 After the cellular and molecular processes,the protein is assembled on the cell membrane. Genomic RNA that budsoff the internal cell membranes is converted to the mature particleforms.43The receptors utilized by humanCoVs typically include 9-O-acetylatedsialic acid by HCoV-OC43 and HCoV-HKU1,344 Two routes are used by CoVs for entering humancells. These routes are categorized as direct delivery of the viralgenome into the cytosol through fusion with the host cell membraneand through endocytosis  and CD147 were recently identified as host cofactorsthat enhancing the entry of SARS-CoV-2 via endocytosis.49 Proteolytic cleavage at the S1/S2 and S2′ sites by host cellproteases is required for the conformational changes of the S proteinand for the viral fusion with the cell membranes. The cell surfaceserine protease transmembrane serine protease 2 (TMPRSS2) and endosomalcysteine proteases cathepsin B and L (CatB/L) are responsible forthe activation of the spike proteins.50 After binding to the ACE2 receptor, host proteases on the cell surfacemediate virus fusion at the level of the plasma membrane or with theendosomal membrane, with subsequent release of the viral genome intothe host’s cytosol.51 Release ofendocytosed virions into the cytosol is usually dependent on the pHof the endosomes, whereas direct entrance of the virions into thecytosol is pH-independent.Coronaviruses enter the host cell through the interactionof theirstructural spike protein with cell surface receptors. The S1 subunitof the viral spike protein binds with its receptor through the receptorbinding domain (RBD), after which fusion with the viral cell membranecommences through the spike S2 subunit.52 However, similar to the SARS-CoV and other CoVs, SARS-CoV-2 mayutilize multiple pathways to gain access into the host cell cytosol.57 To date, 11 clinically approved generic drugs have been identifiedas potential candidates for blocking the routes of entry of SARS-CoV-2,including direct fusion with the cell membrane.Recently, it has been proposed thatSars-CoV-2 employs clathrin-mediatedendocytosis as the mechanism for cell entry.458 However, in some cases,viruses can evade the immune system and spread to other tissues, includingthe respiratory system, central nervous system, cardiovascular system,gastrointestinal system, liver, and kidney, where they induce othertypes of pathologies , IL-6, IL-12, interferons, andtumor necrosis factor (TNF)-α. This “cytokine storm”ultimately leads to an acute respiratory distress syndrome (ARDS),which is characterized by endothelial cell dysfunction, damage ofthe vascular barrier, capillary leakage, and diffuse alveolar damage.66 A severe formof ARDS with low oxygen saturation levels and respiratory failureis the leading cause of mortality for SARS-CoV-2.Other factors involved are enormous oxidative/nitrosativestressfollowing the entrance of the virus, with the occurrence of apoptoticcell death and necrosis.67 or corticosteroids such asdexamethasone68 are considered promisingtherapeutic candidates for counteracting lung hyper-inflammation.These medications generally improve clinical outcomes.The molecularmechanism involved in the pathogenesis of SARS-CoV-2is characterized by cytokine dysregulation. Accordingly, cytokineblockers such as tocilizumab, sarilumab, and siltuximab monoclonalantibodies4.269 It has been demonstratedthat HCoV-OC43 RNA has the potential to cause persistent infectionin human CNS cells for at least one year in a murine model of acuteviral encephalitis.70 In murine CNS, neuronswere the main target of viral infection; the neurons were degeneratedvia programmed cell death.71 The S glycoproteinof the virus plays a major role in the neurodegenerative mechanism.72 Infections involving HCoV-229E, HCoV-OC43, SARS-COV-1,and SARS-COV-2 have been identified in various human neurologicaldiseases, such as Parkinson’s disease, multiple sclerosis,and acute disseminated encephalomyelitis.75 To date, therehave been no reports on the presence of HCoV-HKU1, HCoV-NL63, or MERS-CoVin the central nervous system of humans. However, several studieshave shown that neurological symptoms are associated with HCoV-HKU1,HCoV-NL63, and MERS-CoV.77 SARS-CoV-2 was detectedin capillary endothelial cells in the frontal lobe tissues obtainedfrom the post-mortem examination.78 Accordingto that report, viral infections that cause neurodegenerative diseasescan impair the function of the blood brain barrier and illicit a systemicinflammatory response.79 The systemic inflammationtriggered by coronavirus infection may cause neuroinflammatory reactionsthat increase susceptibility of the infected individual to neurologicaldisorders.58 Infection of the central nervoussystem may expedite the progression of neurodegenerative diseasesin at-risk individuals.Detection of CoVsRNA in human brain samples indicates that theseviruses are neuroinvasive and neurotropic, with the capability ofcausing CNS diseases.4.380 A recent case study identified SARS-CoV-2 RNA in a stool specimen,where the virus utilized ACE2 receptor for the entry into the cells.81 Indeed, ACE2 expression correlates with neutralamino acid transporter B0AT1 (SLC6A19) expression in the gastrointestinaltract, which increases the susceptibility of an individual to CoVinfection.82The relationbetween respiratory infection and the gastrointestinal tract has notbeen completely understood. Patients with respiratory infections typicallyhave intestinal dysfunction. This is indicative of the crosstalk betweenthe gastrointestinal tract and the lung.4.484 The mechanism of severe myocardial damage caused by CoV infectionmay be related to the ACE2 cell surface receptor. Indeed, ACE2 isextensively expressed both in the lung and in the cardiovascular tract.Hence, ACE2-related signaling pathways may have a key role in heartdysfunction.85 Other suggested mechanismsof myocardial injury include a cytokine storm that is triggered byan imbalance between type 1 and type 2 T-helper cells, and respiratorydysfunction and hypoxemia caused by SARS-CoV-2 that result in damageof the myocardial cells.86Myocardialdamage caused by CoV infection increases the complexity of patienttreatment. Recent studies reported that MERS-CoV and SARS-CoV-2 cancause severe myocarditis and heart dysfunction.4.587 In addition, SARS-CoV-1RNA was demonstrated in hepatocytes by reverse transcription-polymerasechain reaction (RT-PCR).88 Because theACE2 receptor is abundantly expressed in the endothelial cells ofliver, it has been proposed that SARS-CoV-1 utilizes this receptorfor cell entry.89 Both liver cells andbile duct epithelial cells express ACE2 receptors.90 However, bile duct cells express more ACE2 receptors thanliver cells. Because bile duct epithelial cells play an importantrole in liver regeneration and immune response,91 it has been suggested that liver damage that occurs inCoV patients is attributed to the damage of bile duct cells and notthe virus infection.Post-mortem examinationof patients infected by SARS-CoV identified the presence of a largenumber of virus particles in the lungs, liver vascular endothelium,and parenchymal cells.96 A more recent study reported that liver dysfunction in patientswith severe SARS-CoV-2 infection was significantly more extensivethan that patients with mild SARS-CoV-2 infection only.81 In those patients with severe SARS-CoV-2 infection,the levels of liver enzymes such as alanine aminotransferase, aspartatetransaminase, and gamma-glutamyl transferase are considerably high.97Liver enzymes and bilirubin levels increasedin patients with MERS-CoV infection, whereas albumin levels decreased.98 The antibiotics, antiviral medication, and other drugs used forthe treatment of CoV infection probably cause liver dysfunction.100Patients infected with coronavirus whohave other liver comorbiditiessuch as hepatitis B virus (HBV) or hepatitis C virus (HCV) infectionsare more susceptible to liver damage and the manifestation of acutehepatitis. This may be attributed to the promoted replication of thehepatitis virus during CoV infection.4.6101 It is well shown that ACE2 receptorsare not only expressed in the lung, heart, liver, and brain but arealso present in the kidney.103 Thus, the virus canutilize this receptor for entry into the kidney. Patients sufferingfrom SARS-CoV-2 infection have been reported to have a higher frequencyof renal and kidney abnormalities.104Studies have shownthat CoVs (SARS and MERS-CoV) can attack the kidney and cause acutekidney injury.5105 There is growing interestin virus detection through the use of molecular-based techniques.These approaches have been classified into the amplification or nonamplificationmolecular-based techniques.106The innate immune system providesexcellent defense against viruses,otherwise primary prevention is the only alternative option. For thisreason, diagnosis remains the most effective approach to control virusinfection.107 Fully automated detection systems are generally preferredin medicine. Biosafety issues and time concerns associated with theclinical usage and study of viruses are eliminated with the use ofsuch systems. Despite all the purported benefits associated with thenewly developed molecular techniques, there are still potential restrictionsregarding their accuracy, sensitivity, specificity, and even reproducibility.These restrictions are mainly caused by the genetic inconsistencyof viruses.109 In addition, these assays areexpensive and time-consuming, requiring specific laboratory instrumentsas well as expert human resources.110 Nanomaterialswith unique properties, including optical, electronic, mechanical,and magnetic characteristics, are considered attractive substratesfor biomedical imaging and clinical diagnosis.112113Molecular-basedtechniques are more rapid and sensitive than serologicaltechniques, either as a simple method for the manual detection ofviruses or as a part of highly developed techniques.121 Nanobiohybridplatforms, containing at least one component derived from virus  are conjugatedto various NPs.122 These systems rely onfunctioning of NPs as well as the activity of the conjugated biomoleculesand/or compact multivalent probes for signal transduction.123 These specific NP-based probes are used ina variety of optical, electrical, and electrochemical assays for singleand multiple virus detections.124According to the WHO, the currenttrend of CoV diagnostics is focusedon the development of nucleic acid- or protein-based detection methodologyfor point-of-care testing (POCT).125 More recently, RT-PCR was combined with lateralflow immunoassay for rapid detection of MERS-CoV.126 Nucleic acid testing can also be combined with the lateralflow assay. For example, a multiplex colorimetric paper-based analyticaldevice was developed using AgNPs as a colorimetric substrate to detectthe DNA associated with MERS-CoV infection.127 Another system was developed using self-assembled nanostructurethat consisted of AuNPs and quantum dots.128 This platform was used as an immunosensor for the detection of Aviancoronavirus (IBV) infected birds. Nanonested PCR was employed withAgNPs to distinguish between the variant and the classical strainsof porcine epidemic diarrhea corona virus.129 In another study, a method was developed for detection of IBV usingmagnetoplasmonic NPs and zirconium-quantum dots conjugated with IBVantibodies.130 Notably, there was no reactionbetween the magnetoplasmonic NPs and Zr-quantum dots until the targetedvirus was added.131 Compared with conventionalanalysis, this immunosensor possesses remarkable advantages, includinghigher sensitivity, faster analysis and accuracy comparable to enzyme-linkedimmunosorbent assay. In 2019, a range of signal amplifying techniqueswere introduced, including thermal imaging and assembly of multipleAuNPs, for improving the lateral flow readout signals for the detectionof MERS-CoV  protein.MERS-CoV 4.132133 Monoclonal antibody–colloidal gold conjugates wereutilized as tracers during the preparation of ICS. The assembled ICSwas identified as a specific test for IBV antigens, compared to RT-PCR.133 Considering that RT-PCR is an expensive technique,the AuNP-ICS method appears to have the potential for rapid detectionof different IBV strains in chickens.An immunochromatographicstrip (ICS) was introduced for the detectionof IBV in infected chickens based on the use of IBV-specific monoclonalantibodies against S glycoprotein and N proteins.134 In the lateral flow assays, a paper strip is coated withAuNP–antibody conjugates in the first line and with captureantibodies in the second. A urine or blood sample is placed on thestrip, while the proteins of interest are placed on the membrane.135 The viral antigens bind to the coated AuNPsin the first line as the sample runs through the membrane by capillaryaction. When the antigen/AuNP–antibody complex flows throughthe strip, it is immobilized by the capture antibodies in the secondline and a colored line appears. The color of the complex (blue) isdifferent from the color of the NPs (red) because of plasmon effect.Although this kind of assay shows 100% specificity for IgM and IgG,the clinical sensitivity and accuracy are different . Detecting both IgM andIgG yields a clinical sensitivity of 82%.135Lateral flow detectionof SARS-CoV-2 antigen has been used to improveCOVID-19 diagnosis as a point-of-care approach.136 In that study, fluorescence of thequantum dots was quenched upon their binding with AuNPs in the vicinity.Fluorescence was recovered by neutralizing SARS-CoV2 antibodies thatcompete with ACE2–AuNPs or blocking the binding of quantumdot–spike protein RBD to the ACE2–AuNPs. The in vitrobioimaging results demonstrated the potential ability of quantum dot–RBDinternalization via dyamin/clathrin-dependent receptor-mediated endocytosis,with high affinity to the ACE2 extracellular domain. This platformis a promising biosensor for facile, rapid, and high-throughput cell-basedscreening of SARS-CoV-2 infection.An energy transfer system has recently been developed usingrecombinantspike protein receptor binding domain (RBD) conjugated to fluorescentquantum dots, AuNPs, and cells with green fluorescent protein taggedACE2 receptors (ACE2-GFP) for facile monitoring of viral spike protein–ACE2interaction 5.136 In 137 This biosensingplatform could recognize surrounding alteration on their surface andprovide ultrasensitive sensing and low-noise detection. In addition,it could distinguish the SARS-CoV-2 antigen from the MERS-CoV antigen.It is a potential device for rapid and highly sensitive detectionof CoVs from clinical samples.Another research groupcreated an advanced field-effect transistor(FET) biosensor platform based on graphene sheets with a specificantibody against SARS-CoV-2 spike protein 6.137 Thi2 nanosheets)and quantum dots were employed for the detection of a range of coronaviruses.138 Conjugating nanomaterials with colorimetric,electrochemiluminescence, immunosensing, photoluminescence, and chiroimmunosensinghave also been considered as potential substrates for the detectionof coronaviruses. Electrochemical devices appear to be a good alternativefor the detection of new strains of coronaviruses because of theirsuperior ability to combine with nanomaterials.138 Using nanomaterials in this aspect decreases the time ofanalysis and increases sensitivity. This strategy opens new vistasin designing better systems with higher performance in the future.Metal NPs  to detect antibodies against specificinfections such as human immunodeficiency virus (HIV) infection throughcolorimetric detection.140 This platformshowed 100% clinical sensitivity and 87% specificity for HIV detectionin 96 patients in Rwanda. Microfluidics may be modified further forthe detection of coronavirus RNA or protein.Microfluidic devices incorporated as organ-on-a-chip are consideredanother point-of-care system.66.1169 In 2014, a research group patented a mixture of silver colloid,titanium dioxide (TiO2) NPs and a dispersion stabilizerwith antibacterial, antifungal, and antiviral behavior.170 The platform offers antiviral activity againstCoVs such as porcine epidemic diarrhea virus (PEDV) and swine transmissiblegastroenteritis virus (TGEV). When the platform concentration is dilutedby 1000-fold, virus growth is inhibited at a rate of 99.9 and 93.0%for PEDV and TGEV, respectively. This activity was reliant on theplatform concentration, which means that the usage dose has to bein tune with the virus in which the platform is designed to inhibit.170Nanomaterialshave been introduced as antiviral agents or drug deliveryplatforms for combating CoV infections.171 These nanomaterialsincluded AgNPs, two Ag nanowires with mean lengths of 60 and 400 nm,and silver colloids. Silver NPs and the two types of Ag nanowiresprotected the testicle cells against TGEV infection and reduced thenumber of apoptotic cells. In contrast, the silver colloids were notcapable of inhibiting cellular entry by TGEV.171 Graphene oxide–silver (GO–Ag) nanoconjugatesthat possess antiviral activities against nonenveloped and envelopedviruses were developed by other researchers.172 Different dilutions of GO–Ag solution were incubated withdiluted solutions of feline coronavirus. The supernatant was analyzedusing a virus inhibition assay after removing the GO-Ag pellets. TheGO-AgNPs were able to detect nonenveloped and enveloped viruses bybinding of the AgNPs to the negatively charged sulfur groups of theviral proteins, whereas pristine GO inhibited only enveloped virusesat noncytotoxic concentrations.172In the same year, the induced immuneresponses of four silver nanoconjugates on TGEV-infected swine testiclecells were investigated.173 Diphyllin is a vacuolar ATPase required forendosomal acidification inhibition in Felis catus whole fetus-4 cells.The inhibitory behavior of diphyllin against FIP was enhanced by generatinga diphyllin nanocarrier with poly(ethylene glycol)-block-poly(lactide-coglycolide). Diphyllin NPs demonstrated antiviralactivity; even a high dosage of the NPs was tolerated by mice.173 Although this system was not a candidate forpreparing vaccines, the study verified the efficacy of nanoformulationsagainst coronaviruses.A diphyllin-based therapeutic device was developed for the treatmentof feline infectious peritonitis (FIP) caused by feline coronavirus.N-(2-hydroxypropyl)-3-trimethylchitosan (H-HTCC) to produce nano/microspheres (NS/MS) for adsorbingcoronaviruses.174 The copy number of viralRNA decreased when H-HTCC-NS/MS was added to the viral suspensions.The result is indicative of a good correlation between virus concentrationand the amount of added biomaterial.174 In another novel therapeutic approach, Ag2S nanoclusterswere fabricated for restraining the proliferation of PEDV in treatedVero cells , the results derived fromSARS-CoV may be extrapolated to SARS-CoV-2 (178Ribonucleic acid interference (RNAi) mediatedby small interferingRNA (siRNA) is an effective strategy to inhibit the replication ofRNA viruses. Antiviral siRNA therapy offers several advantages comparedto conventional antiviral drugs and vaccines. These advantages includerapid action with high specificity and efficacy at different viralstages, the use of a less amount of siRNA to reduce viral RNA, andhigh homology of siRNA with cognate viral RNA.RS-CoV-2 8A.178179 A research group reportedthat the use of expression cassettes (plasmid-mediated siRNAs) thatproduced six antiviral RNAi activators could target specific sitesof the viral genome. Pretransfection of Vero cells with the siRNA-expressingplasmids pSR02 and pSR03 prior to the infection of those cells withSARS-CoV resulted in blocking the replication of the ORF1b sequenceof the virus genome.180 Targeting the Ssequence effectively inhibit viral infection and replication becausethe S gene is a good target in SARS-CoV.181 RNAi activators that target both S and ORF1b regions of the viralgenome have been investigated as the potential drug candidates.183 Based on these valuable results derived from the use of RNAi againstSARS-CoV, gene therapy via RNAi may revolutionize the treatment ofCOVID-19.184 The therapeutic potentialof RNAi in combating MERS-CoV has been investigated by using two siRNAs,Smad7-1 and Smad7-2, to knockdown MERS-CoV in both human lung andkidney cell lines. It was found that Smad7 effectively inhibited viralreplication and infection in host cells.101Several recent studies have found that RNAi iseffective againstSARS-CoV.185 Application of siRNA is typically hamperedby rapid enzymatic degradation of the siRNA, fast clearance and inabilityof SiRNA in entering cells.186 These challengesare mostly due to unstable negatively charged siRNA bases that stimulateunwarranted immune response, and random insertion of the siRNA intochromosomes that results in gene dysfunctions.187 These restrictions may be overcome by using nontoxic, biocompatiblenanocarriers prepared from polymers, lipids, hybrid (polymer/lipid)NPs, nanohydrogels, silica, dendrimers, iron oxide NPs and AuNPs.190 Among these, lipids and polymers are considered promising platformsfor siRNA delivery because of their highly biocompatible and biodegradablenature. For example, poly(lactic acid), polycaprolactone, poly(glycolicacid) and their copolymers have been approved by the United StatesFood and Drug Administration for targeted siRNA delivery in vivo.192Although specific targeting of the viral genome sequence isthestrength of antiviral siRNA therapy, targeted delivery of siRNA intoa cell with inadequate endosomal escape is another potential approach.193 Nanocarriers preserve the encapsulatedsiRNA from degradation by serum nucleases, prolong their circulationand promote their access to destined sites.194 Polycationic lipids or polymers maintain their low endosomal pHby increasing influx of protons and water. This causes the endosomesto rupture and release the loaded therapeutics into the cytosol.195 Delivery of antiviral siRNA through commerciallyavailable cationic lipid structures such as oligofectamine, lipofectamine(Invitrogen), lipofectin, TransIT TKO (Mirus), and RNAifect (Qiagen)have demonstrated promising results.195 Poly(lactic-co-glycolic acid) (PLGA), lipid, andpolymer–lipid nanocarriers are suitable for loading of inhalableantiviral siRNA as well as for aerosol-based pulmonary delivery ofantiviral siRNA.196 Cholesterol-conjugatedlipid nanoparticles (LNPs) have also been developed for the deliveryof an mRNA vaccine against SARS-CoV-2.197 Histidine-lysine copolymer and spermine-liposome conjugate-basednanocarriers have also been approved for siRNA delivery to targetspecific sequences in the SARS-CoV genome.198 Coronavirus-infected mice that were treated with intranasally deliverednanoformulated antiviral siRNA showed very positive effects. Consideringthese successful achievements, the use of cationic-liposomal encapsulatedantiviral-siRNA and their aerosol formulation appears to be a reasonabletreatment for SARS-CoV-2 infection.199 Alipid/polymer-based nanocarrier modified with functional molecules was effective in delivering siRNA totarget sites through intranasal or intratracheal administration viaan inhaler  is a good alternativefor functionalization of nanocarriers and the subsequent deliveryof therapeutic siRNA to lung cells and other organs that express thesemarkers. The surface of nanocarriers may also be functionalized withpolyethylene glycol and pH-sensitive histidine-lysine peptide forprolonged circulation and endosomal release of siRNA to the cytosolfor inducing the RNA interference pathway.200 Activation of the RNA interference pathway results in cleavage ofthe viral RNA at the targeted site, which is critical for combatingviral infection.Lipid-based NPs, includingsolid-lipid NPs, nanostructured lipids,and liposomes, are also suitable for the preparation of siRNA deliverysystems. inhaler 8B.200 Th6.3Immunotherapy-based NPs have gainedattention as a highly effectivetreatment modality for combating infectious diseases. However, thereare still challenges associated with increasing therapeutic efficiencyand reducing side effects. Understanding the function of the immunesystem against infection and the possible approaches to modulate immunityare essential steps toward the design of effective immunotherapy.6.3.1201 In the eventof an acute infection, other immune cells, including natural killer(NK) cells, alveolar macrophages, monocytes, and neutrophils, areactivated. This produces a large amount of pro-inflammatory cytokines-α, interleukin (IL)-1β,and IL-6), resulting in a condition known as the cytokine storm thatseverely impairs the respiratory epithelial cells.5 The innate immune cells use pattern recognition receptors(PRRs) such as retinoic acid-inducible gene I-like receptors, Toll-likereceptors, and nucleotide-binding and oligomerization domain-likereceptors to detect PAMPs and generate an appropriate immune response.202 Subsequent interactions between PAMPs and PRRsstimulate phagocytosis by macrophages and dendritic cells and induceintracellular molecular pathways to express pro-inflammatory cytokines, IFNα/β; and type IIinterferon (IFN-II), IFN-γ) and chemokines . The IFN-I blocks the replication of viruses through multiplepathways.203 The infected cells that expressmajor histocompatibility complex I induce NK cells to produce IFN-γand stimulate apoptosis via antibody-dependent cellular cytotoxicity.204The immune responsesto CoVs include innate and adaptive immunity.When CoVs encounter the first line of immune defense , the pathogen-associated molecular patterns (PAMPs)on the virus surface alert the innate immune cells to the presenceof the invading molecule. This results in the release of type I interferons(IFN-α/β).+/CD8+ T cells.206 In this infection,macrophages and dendritic cells have an essential role in mountingspecific immune responses. These cells remove virus particles throughphagocytosis and IFN-I secretion, with subsequent priming of the adaptiveimmune responses.207 In additions, IFN-Iinhibits the replication of viruses through upregulation of interferonstimulated genes, including protein kinase R (PKR) and 2′-5′-oligoadenylatesynthase (OAS)/RNase L.203 These importantcomponents of the protein synthesis machinery block the synthesisof proteins via phosphorylation of OAS/RNase L and eukaryotic initiationfactor 2 subunit-α (eIF2α), resulting in degradation ofthe viral ssRNA and impairment of viral replication.208 The IFN-I promotes CD8+ T cell priming, inducesB cell activation and antibody production, and eventually stimulatesNK cells and macrophages to halt viruses. Several studies have reportedthat MERS-CoV expresses NS4a protein to block the activation of PKRand OAS/RNase L in the innate immune responses.211 The response of the immune system to SARS-CoV-2 infection is shownin 212 An invitro study reported that IFN-α could restrain SARS-CoV infection.213 A more recent study also indicated that pretreatmentof cells with IFN-I resulted in a significant decrease in SARS-CoV-2replication. These initial findings suggest that IFN-1 possesses antiviralactivity against SARS-CoV-2.214 However,more clinical trials are required to validate these findings.In SARS-CoV-2 infection, extensive productionof antibodies was observed along with reduction in CD46.3.2216 Antibodies recognize and mediate the killing of the virus-infectedcells via several pathways, including phagocytosis, opsonization,neutralization, and activation of the classical complement pathway,as well as mediating antibody-dependent cellular cytotoxicity.217 As such, the virulence of virus and the hostimmune response should be balanced to successfully overcome the viralinfection. Although the host’s inflammatory responses in theearly stages of infection is essential, the severe inflammatory responsesat the late stages of the viral infection aggravate the clinical manifestations.218 For this reason, immunotherapy strategies thatenhance viral clearance and minimize the hyper-inflammatory responsesshould be used to overcome coronaviruses infection.219 Immunotherapy against SARS-CoV and MERS-CoV infectionsis classified into three approaches: passive antibody therapy, interferonα/β and IL-6 receptor inhibition  through the use of immunosuppressive drugssuch as tocilizumab and chimeric monoclonal antibody such as siltuximabcan overcome cytokine storms and reduce the clinical manifestationsin SARS-CoV-2 patients.228Cytokines are other potential targets for efficient immunotherapyof coronaviruses. Among the cytokines, IL-6 is considered more importantin the treatment of SARS-CoV-2. This is because overexpression ofIL-6 is associated with the severity of inflammatory cytokine storm.229 Notably,all immunotherapy efforts against SARS-CoV-2 mostly involve the useof polyclonal antibody via plasma therapy, polypeptide hormone forT cell maturation, neutralizing antibodies, ACE2 immunoadhesin, immunoglobulins,and monoclonal antibody against IL-6.230 In spite of the extensive attempts in the development of monoclonalantibody-based passive immunotherapy for combating CoV infections,no monoclonal antibody is available to date. The major limitationis that large-scale production of monoclonal antibodies is difficult,expensive, and time-consuming.231 Designingand developing advanced platforms and materials are essential in providingimmunotherapy at a reasonable cost in a short time period.The useof the described immunotherapy approaches, alone or incombination with other drugs, has been proposed for treating patientswith SARS-CoV-2 infection.232 For example, nanoparticulate forms of antigens and other immunomodulatoryagents can modulate the function of immune components through enhancingmultivalent receptor cross-linking, regulating intracellular processing,inducing cytosolic delivery, targeting the innate immune system, andreducing the toxicity associated with immunomodulators.233 In additions, nanomaterials have the potentialto incorporate several antigens on their surface for more effectiveactivation of the immune system. Thus, nanomaterials are not onlytherapeutic carriers but may possess immunomodulatory properties themselves,acting as potential immune adjuvants. To date, an extensive rangeof nanomaterials such as dendrimers, liposomes, carbon nanotubes,polymer-based materials, and inorganic NPs have been investigatedas potential platforms for immunological applications.234 Nanomaterials such as PLGA and liposomes canactivate CD8+/CD4+ T cells and promote antigencross-presentation for effective antigen delivery.236 Moreover, inorganic NPs such as AuNPs can interact with dendriticcells, promoting the expression of pro-inflammatory cytokines , and the down-regulationof anti-inflammatory factors -β1and IL-10).238 Gold nanoparticles also activateT cells-related immune responses and increased the phagocytic activityof dendritic cells. Despite the progress of experimental applicationof nanomaterials in immunotherapy, there are relatively few fundamentalinvestigations on the use of NP-based immunotherapy against CoVs.Nanoparticleformulations are promising platforms for overcomingthe hurdles associated with immunotherapy.6.4239 Some of the current vaccines utilize either delivered or expressedviral proteins to induce neutralizing antibodies against CoVs. Theseantibodies inhibit viral entry by binding to the M, E or S proteinsof CoVs.240 The use of nanobased therapeuticagents against different types of CoVs has been perceived as a potentialsolution based on the immunostimulatory effects of NPs.241Because of their specificity and capacity to induce immune memory,vaccines are the preferred defense tools against infectious diseases,compared to chemotherapeutical drugs.242 The useof this antigen-colloidal gold complex resulted in the activationof macrophages and immunity against TGEV, with induction of IFN-γproduction and higher titers of neutralizing antibody in vaccinatedanimals. Proliferation of T cells was amplified ten-fold followingimmunization with the antigen-colloidal gold complex, compared withthe free antigen response. On the basis of this result, virus-conjugatedAuNPs were suggested as a potential antiviral vaccine.242Gold nanoparticles conjugated with TGEVwere used for stimulating the protective immune response against CoVin immunized mice and rabbits.243 Theuse of this NP-based vaccine against MERS-CoV induced CD4+ T cells and promoted the production of TNF-α and IFN-γ(244 Usinga recombinant adenovirus serotype 5 that encodes the MERS-CoV spikegene (Ad5/MERS) and spike protein NPs, female BALB/c mice was immunizedthree times with the prime-boost vaccination. The homologous immunizationwith spike protein NPs successfully induced higher antibody titers.However, Th1 immune response was not generated by spike protein NPs.Only the Th2 immune response was elicited with the induction of neutralizingantibodies. A heterologous one-stage Ad5/MERS prime and two-stagespike protein NP boost appear to be more effective than the homologousprime-boost regimen in providing more durable immunogenicity and balanceof Th1/Th2 responses.244Ribonucleic acid conjugated to ferritin-basedNPs have been proposedas a potent molecular chaperone.nd IFN-γ10. In an7246 To date, developinganti-COVID-19 drugs have encountered challenges because of their sideeffects to the lung and heart. A smart technology is therefore requiredfor the design and fabrication of rational drugs that only targetSARS-CoV-2 with minimal side effects.247 Drug repurposing is an effective drug discovery strategy based onthe use of existing drugs. Such a strategy shortens the time and reducesthe cost compared to de novo drug discovery. In silico pharmacology performed on a computer or via computer simulationis a smart, revolutionary technology for evaluating approved medicine,reducing the regulatory costs of innovation and decreasing the timefor marketing of biomedical products. Such a “virtual”process is indispensable in contemporary drug discovery research fortranslating drugs into clinical trials.249 The combination of in silico strategy and large drug-related databasesfacilities the selection of appropriate repurposed drugs by screeningtheir side effects on different organs. Drug-repurposing strategieshave recently been performed by computational modeling on the interactionand mechanism of potential drugs with the host cells and SARS-CoV-2250 Computer modeling offers a platform for visualassessment and analysis of the molecular mechanisms involved in theentrance, replication, and transcription of virus molecules as wellas their interactions with host cells, immune response, and the potentialmechanisms of cell recovery.Translating archived knowledgeacquired from the laboratory intoclinical trials is a crucial and challenging stage for safe and tangiblecombat against COVID-19.251 Inthis approach, RNA sequences were collected from the Global Initiativeon Sharing All Influenza Data (GISAID) database to investigate the3D protein sequences and protein–ligand interactions via homologymodeling. Drug screening was performed without using docking or moleculardynamics. This modeling successfully recognized chemical ligands  and peptide drugs  from the databases to aid scientistsin identifying molecules that can combat SARS-CoV-2 in a shorter timeperiod.Another research group investigateda deep-learning Dense FullyConvolutional Neural Network (DFCNN) model for screening establisheddrugs against SARS-CoV-2 infection.252 In this work, phylogeneticanalysis of 15 human CoV whole genomes with coronavirus infectionwas performed. Using network proximity analyses of drug targets andhuman CoV–host interactions in the human interactome,16 potential anti-CoV repurposable drug candidatessuch as melatonin, mercaptopurine, and sirolimus were identified andfurther validated by enrichment analyses of drug–gene signaturesin human cell lines. In addition, three potential drug combinationswere identified through a “Complementary Exposure” pattern,including (i) sirolimus plus dactinomycin, (ii) mercaptopurine plusmelatonin, and (iii) toremifene plus emodin. The study provides anexcellent role model for the rapid identification of therapeutic drugsfor combating SARS-CoV-2 infections.In another interesting work, an advanced pharmacologynetwork-basedapproach was developed to evaluate a rational drug for effective treatmentof COVID-19 infection 11.252 InCombining data generated on themechanisms of COVID-19 infectionswith in silico models enables virologists, immunologists, clinicians,and computational biologists to collaborate in understanding the accuratemolecular mechanisms of SARS-CoV-2 infection. This approach providesa useful guide for the development of advanced and efficient nanomedicineagainst the COVID-19 pandemic.8253 Nanomaterials possess various physicochemicaland biological benefits. These benefits include reduced particle sizesthat facilitate delivery through natural barriers, larger surfaceareas for higher drug loading, adjustable surface charge to facilitatedrug entry across charged cell membranes, capability to anchor totargeting ligands to increase the specificity of the destined target,superior solubility and pharmacokinetic properties that result inlonger circulation times, better accumulation, controlled/sustainedrelease, and improved efficacy caused by either entrapping drug agentsand protecting them from the physiological environment or surfacemodifications for targeting purposes.256Nanotechnology is rapidly becoming a vivid player in antiviraltherapy for combating coronaviruses. Nanomaterials have been developedspecifically to improve the delivery of biotherapeutics across physiologicalbarriers, thereby resolving the classical challenge of low bioavailability.257 Other challenges associated with the use of nanomaterialsinclude interactions with biological molecules that result in opsonization,phagocytosis by macrophages that reduces their plasma half-life,258 nonspecific absorption which induces apoptosisof the cells that absorb them, and disruption of their cell membranes.259The applicationof nanomaterials as drug carriers, however, isnot free from challenges. One of the most eminent challenge is theirdegradation prior to reaching the target. Nanoparticles, for example,are degraded in the gastrointestinal tract when they are administratedorally. Nanoparticles are not always successful in crossing the mucusbarrier, which results in reduced or nonabsorption.co-glycolide) are close-to-idealcandidates because of their flexibility to uptake various charges,capacity to be fabricated in different shapes and sizes for enhancingthe permissibility of the composition, and reduced clearance to prolongcirculation time.260 Polymeric nanomaterialsare likely to emerge as the materials of choice for the developmentof vaccine and drug carriers for single-dose and needle-free delivery.261An ideal nanocarrier for proficient antiviraltreatment needs topossess several attributes. These attributes include: (1) excellentclinical outcome, as therapeutic devices are required to be effective,available, targeted, safe, and affordable; (2) the nanocarrier needsto improve the efficacy of drug delivery, reduce intake rate and time,decrease side effects, and reduce the cost of therapy; (3) the nanocarriershould possess an appropriate fabrication design that permits targeteddrug delivery in a sustained released manner. Hybrid nanosystems havethe potential to meet the requirements for nanomanufacturing and shape/sizeconfigurations. In addition, the nanomaterials used for fabricatingthe designated compositions should be biodegradable, biocompatible,and nontoxic. In this regard, polymers offer tremendous potentialfor chemical surface modifications. More complex challenges are associatedwith nanocarrier shape because this property is associated with NPsize and surface charge. Polymer-based nanomaterials such as polyethyleneglycol and poly.263 For cyclodextrin drug deliverysystems such as hydroxypropyl beta-cyclodextrins (HPβCD), theuse of carbon-based nanosheets may overcome formulation challengesof antiviral drugs by improving solubility and bioavailability.265 Likewise, they may be used as safe and efficient adjuvants in vaccinesfor coronaviruses. Metal NPs such as AgNPs, AuNPs, MNPs,and their related compositionsmay be used as alternative candidates for the delivery of therapeuticagents against CoVs.In the grand scheme of things, the applications of nanoplatformsfor the detection of human coronaviruses have yet remained unresolvedfor nanotechnology researchers. Colorimetric sensing, electrochemiluminescence,immunosensing, photoluminescence, and chiroimmunosensing, as wellas electrochemical sensors, are potential techniques to detect coronaviruses.Various nanobased vaccines have demonstrated the potential to inducea more potent immune response. However, further investigations onthe interaction of virus particles with host cells are required totackle the application of smart NPs against the mutated versions ofhighly contagious SARS-CoV2 13.267 Although more than 250 other vaccines are in various stages of development,the emergence of new SARS-CoV2 variants268 with possible highly transmissibility demonstrate the urgency ofdeveloping new vaccine formulations with high effectiveness.As of February 2021, eight COVID-19 vaccines based on differenttechnologies have been approved or authorized for emergency use. Theyare the mRNA vaccines BNT162 from Pfizer/BioNtech and mRNA-1273 fromModerna, the chimpanzee adenovirus-based AZD1222 (Covidshield) vaccinefrom Oxford-Astra Zeneca, the Ad26-based viral vector vaccine fromJohnson &Johnson, the virus-inactivated Covaxin vaccine from IndianBarhat Biotech, the CoronaVac vaccine from Sinovac Biotech, China,and the human adenovirus-based Sputnik V vaccine from the GamaleyaNational Center of Epidemiology and Microbiology, Russia.