Expression of circular RNAs is known to be deregulated in cancer. Here the most comprehensive set of differentially expressed RNA circles in medulloblastoma compared to cerebellum is provided. Additionally, fusion RNAs are also identified in both cancerous and normal cerebellar tissue. Some of the fusions detected in medulloblastoma are generated by genomic rearrangements that link different genes. However, fusion RNAs are also detected in normal cerebellum. In fact, there are cases where the same fusion RNA is also found in medulloblastoma. This observation underscores that the formation of fusion transcripts may not be limited to chromosomal events but could also result from mechanisms that act at the RNA level. These include read-through transcription of neighboring genes and intermolecular splicing of pre-mRNAs from different genes Importantly, these RNA “recombination” events occur not only in normal but also in cancerous tissue.Background. The cerebellar cancer medulloblastoma is the most common childhood cancer in the brain. Methods. RNA sequencing of 81 human biospecimens of medulloblastoma using pipelines to detect circular and fusion RNAs. Validation via PCR and Sanger sequencing. Results. 27, 56, 28 and 11 RNA circles were found to be uniquely up-regulated, while 149, 7, 20 and 15 uniquely down-regulated in the SHH, WNT, Group 3, and Group 4 medulloblastoma subtypes, respectively. Moreover, linear and circular fusion RNAs containing exons from distinct genes joined at canonical splice sites were also identified. These were generally expressed less than the circular RNAs, however the expression of both the linear and the circular fusions was comparable. Importantly, the expression of the fusions in medulloblastoma was also comparable to that of cerebellum. Conclusions. A significant number of fusions in tumor may be generated by mechanisms similar to the ones generating fusions in normal tissue. Some fusions could be rationalized by read-through transcription of two neighboring genes. However, for other fusions, e.g., a linear fusion with an exon from a downstream gene joined 5′ to 3′ with an exon from an upstream gene, more complicated splicing mechanisms, e.g., trans-splicing, have to be postulated. Medulloblastomas are malignant cerebellar tumors occurring in infants, children and young adults. Proper diagnosis encompasses magnetic resonance imaging, with the treatment options including resection, chemotherapy, and craniospinal radiation ,2,3. DesExons are usually joined in a 5′ to 3′ direction giving rise to mature linear RNA molecules. However, in recent years the possibility that a 3′ exon is back-spliced to a 5′ exon has gained attention. This generally gives rise to a circular RNA containing these exons, and is particularly stable, since it cannot be attacked by exonucleases ,7,8,9,10There is also evidence that linear RNA molecules can be produced where a 3′ exon is found spliced upstream of a 5′ exon. The original observations are over twenty years old ,24, but There are also data supporting rare events that splice together transcripts from different genes in the same genomic locus ,27,28. MHere we analyzed, via RNA-seq 81, medulloblastoma samples encompassing all 4 subtypes, as well as 5 samples of normal cerebellum. Implementation of RNA isolation protocols that do or do not enrich for circular RNAs and of various bioinformatic pipelines resulted in the identification of not only RNA circles encompassing exons from single genes but also of linear and circular RNAs encompassing exons from different genes.https://cbtn.org) (accessed on 1 March 2022), and 5 normal cerebellum RNA samples were purchased from Takara , Amsbio  and BioChain   .The RNA samples were subjected to poly(A) and circular RNA enriched library preparations as described previously . The conmRNA detection: reads from the poly(A) enriched preparations were aligned to the Gencode GRCh38 reference genome assembly using the STAR (v2.5.2) aligner. Counts were assigned to genes using featureCounts (v1.5.1).Circular RNA detection: reads from the circular RNA enriched preparations were analyzed with the CIRCexplorer2 to identify back-spliced junctions and aligned to Gencode GRCh38 using CIRCexplorer2/STAR  with themRNA and circular RNA counts (raw reads) were analyzed with the DESeq2 Bioconductor package (v1.32.0) using the apeglm (v1.14.0) method for effect size shrinkage and log fold change estimations  in R sofFusion detection: reads from the poly(A) enriched and circular RNA enriched preparations were aligned to Gencode GRCh38 using STAR-Fusion  with theDue to the very low expression of the fusion reads, it was impossible to calculate size factors and effectively perform gene expression level comparisons with the DESeq2 method as done with the circular RNA and mRNA datasets. Consequently, we simply pinpointed the fusion transcripts with the highest reads.The NCOA2--TRAM1 and FBXO25--SEPTIN14 fusion transcripts were validated by PCR using oligo(dT) primers as described previously . Primers81 medulloblastoma and 5 cerebellar RNA samples  were firMoreover, circular RNAs differentially expressed in each medulloblastoma subtype compared to the other three subtypes (including cerebellum) were also identified . These lp < 0.05 and |log2FoldChange| > 1 identified 1 down-regulated and 12 up-regulated circular RNAs  and ARL17B (NM_001039083.5), including the exon 3 at the fusion, are identical. Consequently, the pipeline cannot, in principle, discriminate which of these tandem and in the same orientation genes is spliced to the KANSL1 gene . MoreoveWorth noting is that in the three documented linear fusions having exons from a downstream gene spliced to exons from an upstream gene, namely, NCOA2--TRAM1, KANSL1--ARL17A/B, and TFG--ADGRG7, the expression of the downstream gene is higher than the upstream . This isHere the profile of circular and fusion RNA expression in medulloblastoma was interrogated. Using stringent statistical criteria, 367, 185, 188 and 210 circular RNAs de-regulated in the SHH, WNT, Group 3 and Group 4 subtypes of medulloblastoma, respectively, in comparison to cerebellum, were identified. Earlier studies have detected 3 up- and 30 down-regulated circular RNAs in medulloblastoma . Worth nMoreover, using stringent cutoffs, linear and circular fusion RNAs were identified in both cerebellum and medulloblastoma. The presence of fusion RNAs in normal tissue, some of which were also validated by PCR and Sanger sequencing, raises questions as to whether chromosomal rearrangements are involved. In general, DNA in normal cells is considered stable. However, it is still conceivable that, in a small number of cells present in the sample analyzed, such an event might have occurred. This would be in line with the low number of reads for the fusion RNAs in comparison to the much higher number of reads, observed in standard RNA-seq, for the mRNAs of the two genes that are involved in the fusion.On the other hand, there are alternative scenarios that are also compatible with the observed low number of fusion reads. In cases where the genes are in the same locus and in the same orientation some transcripts from the first gene may extend into the second gene. Forward splicing of these read-through transcripts can generate fusions with exons of the first gene spliced to exons of the second gene, as seen in the linear PAUPAR--RCN1 and RPS10--NUDT3 fusions. Moreover, back-splicing of the read-throughs can generate circular RNAs with exons from the second gene spliced to exons from the first gene, as seen in the circular ADAMTSL3--SH3GL3 fusion.However, linear fusions have also been observed from genes in the same locus and in the same orientation, but with exons from the second gene spliced to exons from the first gene, e.g., NCOA2--TRAM1. In these cases, a read-through mechanism can not interpret the observations. Consequently, scenarios that involve intermolecular splicing of transcripts from different genes, i.e., trans-splicing, have to be postulated. Such mechanisms may also rationalize splicing from genes in different chromosomes/loci or in opposite orientations from the same locus ,29,30.www.ncbi.nlm.nih.gov/gene/202559) (accessed on 1 March 2022). In fact, a KHDRBS2 circular RNA, highly expressed in Group 4 medulloblastomas, has been identified in our analysis (In tumor tissues, it is well-known that chromosomal rearrangements can drive tumor growth ,42. Theranalysis . Thus, sOf particular interest is the KANSL1--ARL17A/B fusion, as both linear and circular forms are observed in numerous medulloblastoma samples. In fact, the KANSL1 exon 3 of the fusion is also involved in back-splicing events generating two KANSL1 circular RNAs, the only two KANSL1 circles identified in our analysis . Exon 3 The number of RNA samples in the cerebellar and the WNT medulloblastoma groups is small, at 5 and 6, respectively, and this can impact the robustness of the RNA-seq analysis. Additionally, the abundance and the number of fusion RNAs detected is quite small and this does not permit an effective statistical analysis of differential expression in cerebellum versus the medulloblastoma subtypes. Increasing the read depth, currently at 25 M reads per sample, should provide the means to overcome this limitation. It is also possible that certain fusion RNAs, similarly to certain circular RNAs, may have limited impact on disease development and could simply represent “passenger” molecules, reflecting the stochasticity of the gene expression and splicing processes ,36. EvenHere, de-regulated circular RNAs in the four subtypes of medulloblastoma have been identified. Additionally, linear and circular fusions RNAs have also been observed. For some of these, e.g., PVT1--CASC8, chromosomal events are implicated. However, for a large number of these fusions, a plausible interpretation is molecular mechanisms similar to the ones generating fusions in normal tissue, i.e., read-through transcription and trans-splicing.


Since the outbreak of the coronavirus disease 2019 (COVID-19) pandemic, there have been a few variants of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), one of which is the Omicron variant (B.1.1.529). The Omicron variant is the most mutated SARS-CoV-2 variant, and its high transmissibility and immune evasion ability have raised global concerns. Owing to its enhanced transmissibility, Omicron has rapidly replaced Delta as the dominant variant in several regions. However, recent studies have shown that the Omicron variant exhibits reduced pathogenicity due to altered cell tropism. In addition, Omicron exhibits significant resistance to the neutralizing activity of vaccines, convalescent serum, and most antibody therapies. In the present review, recent advances in the molecular and clinical characteristics of the infectivity, pathogenicity, and immune evasion of Omicron variant was summarized, and potential therapeutic applications in response to Omicron infection were discussed. Furthermore, we highlighted potential response to future waves and strategies to end the pandemic. Three possible explanations have been proposed for the development of the Omicron variant: silent evolution in a population with little sequencing, long-term evolution in one or a few persons with chronic infection, or evolution in other animals especially rodents.3 Notably, the Omicron variant is not a single strain, but evolved into three lineages: BA.1, BA.2, and BA.3. BA.1 was once the most widely prevalent strain in the world; however, BA.2 is gradually replacing BA.1 in several countries, such as Denmark, Nepal, and the Philippines. The transmissibility of BA.3 is very limited, with very few cases, at most a few hundred cases  was first identified in South Africa and Botswana and was reported to the World Health Organization (WHO) on November 24, 2021, as a novel variant. This novel variant, also known as B.1.1.529, spreads rapidly and was classified as a variant of concern (VOC) by the WHO on November 26, 2021.ses Fig. .Fig. 1Thhttps://covid19dashboard.regeneron.com). BA.2 lineage possesses 57 mutations, with 31 in the S protein, of which the N-terminus is significantly different from that of BA.1. The receptor-binding domain (RBD) of the S protein is responsible for binding to the host receptor angiotensin-converting enzyme 2 (ACE2) and has the potential to increase infectivity and mediate escape from vaccine-induced neutralizing antibodies.6 Therefore, mutations located in the RBD of the S protein have attracted significant research attention. BA.1 and BA.2 share 12 mutations in the RBD, including G339D, S373P, S375F, K417N, N440K, S477N, T478K, E484A, Q493R, Q498R, N501Y, and Y505H. S371L, G446S, and G496S were only identified in BA.1, whereas R346K was found in a member of this group, namely BA.1.1. BA.2 possesses two unique mutations in RBD, including S371F and R408S, and shares T376A and D405N with BA.3  and Gamma (P.1) variants, have been suggested to mediate escape from vaccine-induced neutralization.5 The effects of most of the remaining Omicron mutations are unknown; thus, our understanding of the viral behavior and susceptibility of the Omicron variant to natural and vaccine-mediated immunity remains unclear. Moreover, individuals previously infected with other SARS-CoV-2 variants can be reinfected with this new variant.9 A recent study suggested that the Omicron variant is likely to infect individuals recovering from infections by previously prevalent variants.11 This evidence shows that mutations in Omicron evade immunity induced by the previous infection.The Omicron variant has caused global panic and concern owing to its contagious and vaccine-escape mutations. Presently, up to 60 mutations have been identified in the BA.1 lineage, with as many as 38 of these occurring in the spike (S) protein, one in the envelope (E) protein, two in the membrane (M) protein, and six in the nucleocapsid (N) protein  or cathepsin L,13 The binding affinity of ACE2 for the S proteins of Omicron is one of the main factors determining viral infectivity. To date, the results on the binding affinity of the Omicron variant to ACE2 are slightly different, possibly owing to differences in experimental materials or methods. Moreover, the aggregation state of the protein has a significant impact on measurement results.14 Several studies have shown that the binding affinity of Omicron RBD to ACE2 is approximately 1.5–2.8 times that of the wild-type (WT).17 In contrast, some studies have suggested that the binding ability of Omicron RBD to ACE2 is comparable to that of WT.19 Compared with the previously prevalent Delta variant, Omicron RBD exhibits a similar or weaker binding ability to ACE2.19 In addition, the binding ability of Omicron RBD to ACE2 is much weaker than that of Alpha variant, with only one mutation, N501Y, in the RBD.19 Based on the above studies, it can be inferred that the binding ability of Omicron RBD to ACE2 is roughly between that of WT and Delta RBD. S477N, T478K, Q493R, Q496S, and Q498R have been reported to potentiate the interaction between Omicron and ACE2 by establishing new hydrogen bonds or salt bridges with the corresponding sites of ACE219 in addition to N501Y. However, K417N and E484A can cause a significant loss of polar interactions between Omicron and ACE2, offsetting some of the enhanced interactions forged by other mutations.19ACE2 is a major receptor of SARS-CoV-2.20 These findings indicate the zoonotic potential of the Omicron variant, which could contribute to the development of highly infectious variants.Overall, mutations in the Omicron RBD did not affect its receptor recognition and binding to ACE2, and the Omicron RBD can efficiently bind to human ACE2 for host-cell entry. Notably, the Omicron S protein can bind to human ACE2 or ACE2 orthologs of different animal species to enter the target cells.21 The S1 subunit contains the RBD, which binds to ACE2, whereas the S2 subunit contains the transmembrane portion of the S protein, which is responsible for anchoring the S protein to the membrane and mediating fusion of the viral membrane with cellular membranes.23 Cleavage of the S protein at the S1–S2 and S2 sites, mediated by furin24 and type II transmembrane serine protease (TMPRSS2)25or cathepsin L,26 is crucial for viral entry into host cells. Cleavage by TMPRSS2 and cathepsin L at the S2 site mediates two distinct SARS-CoV-2 entry pathways. As TMPRSS2 is present on the cell surface, TMPRSS2 mediates the plasma membrane route of entry, whereas cathepsin L in the endosome mediates the endosomal entry route.22The entry of SARS-CoV-2 into the host cells after binding to host receptors is mediated by the S protein. The S protein is composed of S1 and S2 subunits.27 Recent studies have reported that Omicron spike pseudotyped virus infection was reduced in TMPRSS2 expressing cells, but increased in cells that support endosomal entry, and that the Omicron variant prefers the endosomal entry route rather than the plasma membrane entry route.30 These findings suggest that mutations on the Omicron S protein non-RBD may alter the route of viral entry into host cells, which is associated with a shift in cellular tropism away from TMPRSS2 expressing cells, and explains the faster replication of Omicron in the upper airways than in the lungs, unlike that of other variants32  that have not been identified in previous VOC.35 The basic reproduction number (R0) of the Delta variant was between 3.2 and 8.36 The transmissibility of the Omicron variant is ~3.2 times that of Delta, and the doubling time is approximately three days.38 Generally, BA.2 is ~1.4 times more transmissible than BA.1,40 with a transmission rate of ~13.4% among household contacts, whereas that of BA.1 was 10.3%41 , sneezing, and sore throat.54 Outcomes in the Western Cape province of South Africa were similar, and the risk of severe hospitalization or death was reduced by ~25%.56 Moreover, patients infected by the Omicron variant and identified by the S gene target failure (SGTF) had significantly lower odds of hospitalization and severe disease than those by Delta.57 Several analyses of patients in the UK showed that the risk of hospitalization with Omicron was approximately one-third of that with Delta,59 with similar observations reported in France and Norway.61 In the United States, the percentage of hospitalization, intensive care unit (ICU) admission, receipt of invasive mechanical ventilation (IMV), and in-hospital death were lower during the Omicron pandemic than during the Delta pandemic, and the mean length of hospital stay was considerably shorter.63 Moreover, a high rate of asymptomatic carriage has been observed since the discovery of the Omicron variant,44 which may suggest milder symptoms of the variant.A few real-world studies have indicated that the Omicron variant may be milder than earlier variants. A study of early cases in the Gauteng province of South Africa showed that the hospitalization rate during the fourth wave (Omicron-dominated) was ~4.9%, which was markedly below the rate in waves dominated by Beta or Delta variant, and the probability of severe illness was reduced by 73% in the Omicron-dominated wave.66 In vitro assays have shown that the Omicron variant poorly induced multinuclear syncytia in multiple cell lines52 compared with previous variants.67 Moreover, higher cell viability was observed in Omicron variant-infected cells compared with those infected with previous variants,32 which was consistent with findings in vivo. Infection with the Omicron variant caused limited bodyweight loss, lower viral load in the upper and lower respiratory tracts, and limited lung pathological damage and mortality rates compared with earlier variants in both hamsters or human ACE2 (hACE2) transgenic mice.53 In addition, the Omicron variant was less effective in antagonizing cellular interferon signaling compared with the Delta variant. Moreover, the activation of the NF-κB pathway is less efficient in response to the Omicron variant,68 implying that Omicron may induce slighter inflammatory responses.34Additionally, preliminary laboratory data confirmed the lower pathogenicity of the Omicron variant compared with the earlier variants. The formation of multinuclear syncytia is a significant pathological step in COVID-19 infection, reflective of cell-cell fusion events during viral infection.71 Notably, the proportion of young patients was higher among those infected with Omicron,72 which may result in reduced pathogenicity. In addition, the impact of Omicron is not attenuated by reduced pathogenicity, the healthcare system is still under enormous pressure because of the high transmissibility of the Omicron variant.73The evidence above further confirms that the Omicron variant is less severe than previous variants. However, it is possible that the pathogenicity of the Omicron variant may be underestimated because of rising levels of herd immunity via previous infections and vaccinations.79 Data from the UK showed that BA.2 and BA.1 have similar immune evasion abilities.81 Moreover, a recent study showed that BA.2 can re-infect BA.1 convalescent patients, despite the small number of such cases identified.80The most serious concern about the Omicron variant is its high immune evasion ability. The Omicron variant can escape the immune response established by vaccination or previous infection by other variants, which may result in high transmissibility.85 Unprecedented complexity in mutation patterns can alter antigenicity, invalidating the existing immunity.86 The cryo-electron microscopy (cryo-EM) structure helps to reveal the basis of immune evasion by Omicron. Omicron S-trimer exclusively formed one conformational state with one “up” RBD and two “down” RBDs, while a single-up conformation and all-down conformation were depicted in previous variants.87 Steric clashes, altered interactions at antibody-binding surfaces, and local changes in the spike structure were induced by mutations that interfered with antibody recognition.87There are several mutations in the RBD region and N-terminal domain (NTD) of the Omicron variant, which are the main targets of neutralization.92 Moreover, Ad26.COV2.S, ChAdOx1-S, and Sputnik V, representative of vectored vaccines, failed to trigger effective neutralizing activity against the Omicron variant.95 RNA vaccines, such as BNT162b2 and mRNA-1273, which were proven to be the most effective against the WT strain, were completely ineffective against the Omicron variant in over 50% of individuals.102 In serum samples from individuals who had received two doses of BNT162b2 or mRNA-1273, there was a considerable decrease in the titers of neutralizing antibodies against the Omicron variant compared with the WT.106 Although each study differed due to differences in samples and testing methods, the findings showed that there was a considerable decrease in the neutralizing potency of two doses of RNA vaccines against Omicron. An observational study in the United States showed that the estimated effectiveness of two doses of RNA vaccines against Omicron was only ~30% 1 month after the second dose, with no effectiveness three months after the second dose for BNT162b2 and 6 months after the second dose for mRNA-1273.107 Data on other vaccines against Omicron variants have not been disclosed , LY-CoV016 (marketed as etesevimab), REGN10987 (marketed as imdevimab), REGN10933 (marketed as casirivimab), COV2-2196 (marketed as tixagevimab), COV2-2130 (marketed as cilgavimab) and CT-P59 (marketed as regdanvimab), which block binding of Spike protein to the receptor ACE2.16 Most of these antibodies lost their neutralizing ability against Omicron due to the destruction of the antigenic epitope.126 K417N, E484A, Q493R, and N501Y are the main sites responsible for the evasion.15 The combination of COV2-2196 and COV2-2130 exhibits neutralizing activity against Omicron; however, its neutralization ability against Omicron is 12–200-fold lower compared with that against the WT,127 due to the N440K and G446S mutations.127 However, this combination, especially COV2-2130, has been reported to retain activity against BA.2, perhaps due to the absence of G446S.128 The second group, represented by VIR-7831/S309 (marketed as sotrovimab), rarely competes with ACE2 but recognizes non-RBM epitopes that are conserved within sarbecoviruses, including SARS-CoV.129 S309 shows resistance to Omicron’s antibody escape, with only a two- to threefold decrease in neutralization efficiency for Omicron compared with the WT.127 G339D and N440K are presumed to interfere with the combination of S309 and spike protein due to the proximity of the antigenic site, but the impact is limited.127 BA.2 seems to have a greater negative impact on S309 because of the S371F mutation.128 Despite the presence of the S371L mutation in BA.1, S371F in BA.2 displayed distinct resistance to neutralizing antibodies compared to S371L.128 Brii-198 (marketed as romlusevimab) possesses neutralization efficiency against Omicron, but the neutralization ability is slightly weaker and can be inhibited by R346K.75 In addition, S2X259 possesses considerable neutralizing potency against the Omicron variant,16 but was less effective against the BA.2 strain.128 LY-CoV1404 (marketed as bebtelovimab), which has recently received EUA approval, possesses a high neutralization capacity against all identified variants.130 Structural analysis demonstrated that the epitope of LY-CoV1404 was highly conserved, except for N439 and N501. Fortunately, the N501Y mutation of Omicron does not interfere with its binding capacity.130In this section, we summarized the effect of neutralizing antibodies from vaccines and previous infections against the Omicron variant. The neutralizing ability of therapeutic-neutralizing antibodies against Omicron is weak. Most therapeutic-neutralizing antibodies with EUA approval or at advanced clinical development stages have failed to neutralize the Omicron variant Table .16,75–7768 Moreover, the efficacy of LY-CoV1404 is yet to be validated in clinical studies.Overall, neutralizing antibodies targeting the conserved epitopes of SARS-CoV-2 variants and other sarbecoviruses may have broad prospects for the control of COVID-19 pandemic. Given that SARS-CoV-2 is prone to mutations, developing neutralizing antibodies targeting relatively conserved sites is an effective option to deal with emerging variants, such as Omicron. There are still some doubts about the efficacy of the antibodies mentioned above in treatment. A randomized controlled trial indicated that neither S309 nor Brii-198 showed efficacy in improving clinical outcomes despite the small sample size.131 These drugs have different mechanisms of action, indicating that the drug sensitivity profile of the Omicron variant does not change considerably in response to the drugs. There are only two missense mutations in the replicase–transcriptase complex  of the Omicron variant, which may have little effect on RNA-dependent RNA polymerase-inhibitor drugs, such as remdesivir and molnupiravir.68 However, current research is still at the cellular level, and more clinical results are needed to support these conclusions. Moreover, the efficacy of several drugs is yet to be proven against Omicron. There is an urgent need to develop new broad-spectrum antiviral drugs in response to changing viruses and the current shortage of vaccines and drugs. Drugs that inhibit viral replication are likely to remain effective against the Omicron variant, which is also the mechanism of drugs that inhibit the binding of viruses to ACE2. The ACE2-targeting antibody has been shown to suppress the Omicron variant at the cellular level,132 which is a likely direction of exploration. Boosting the antibody responses using biochemical methods is also a method that could be explored. A recent study showed that an ultrapotent RBD-targeted biparatopic nanobody exhibited enhanced neutralizing activity against Omicron.133 In addition, it has been reported that engineered extracellular vesicles (EVs) enriched with palmitoylated ACE2 (PM‐ACE2) efficiently captured SARS‐CoV‐2 viruses and inhibited their interaction with cell surface ACE2, leading to reduced infection both in vitro and in vivo.134 Furthermore, it has been demonstrated that patients with COVID-19 exhibit impaired IFN responses, especially in patients with severe disease, which might contribute to the limited antiviral response.138 Therefore, targeting antiviral responses could be important in managing SARS-CoV-2. A recent study showed that the N protein of SARS-COV-2 inhibits the activity of MAVS, a key component of antiviral innate immunity, by undergoing liquid-liquid phase separation (LLPS) with RNA, thereby suppressing the antiviral immune response.139 Interestingly, the 246–365 domain of the N protein, which retains the phase-separation ability, is hardly mutated in the currently reported SARS-CoV-2 variants, including Omicron. Thus, targeting the SARS2-N protein LLPS could be a promising therapeutic approach against infection WT and variants of SARS-CoV-2. These potential treatments may help curb ongoing pandemics as well as any future outbreaks , EIDD-1931, ribavirin, favipravir, nafamostat, camostat, and aprotinin.32 making the virus less pathogenic. Routine vaccination or a previous infection cannot provide effective protection against Omicron, and booster vaccination is required. Additionally, only a few neutralizing antibodies are active against Omicron, whereas most antiviral drugs in development are effective against it.The Omicron variant has attracted worldwide attention since its emergence owing to the high number of mutations, which increased its transmissibility and immune evasion capability. Although the binding ability of Omicron to ACE2 is still controversial, it has enhanced transmissibility, making it the dominant species in several regions within a short period of time. Omicron spikes inefficiently utilize TMPRSS2 to enter cells, but mainly rely on the endocytic pathway, which leads to a decrease in replication in the lung parenchyma and an enhanced ability to infect the upper respiratory tract,In the wake of the Omicron variant, it was believed that the effect would be minimal due to herd immunity established by vaccination and infection, and the development of specific drugs. Moreover, the discovery of the Omicron variant indicates that the attempt to end the COVID-19 pandemic might be hindered by the tendency of the virus to mutate. The major research focus should be the development of vaccines and drugs against possible variants of the virus.142 In naive mice immunized with Omicron-matched vaccines, high levels of potent antibodies against Omicron were produced, but their ability to inhibit previous variants was limited.143Since the outbreak of the Omicron variant, several studies have been performed to improve our understanding of the mechanism and characteristics of the variant. Currently, vaccines, social distancing, and specific drugs are still effective means of resisting Omicron. Homologous or heterologous boosters and new vaccines against Omicron, and possibly new variants, have been proposed to improve protection in response to vaccine failure. However, early animal experiments have shown that Omicron-targeted vaccines were not more effective than booster jabs of already developed vaccines against the variant, although they all generated broad antibody responses to all variants, including Omicron.144 indicating that the occurrence of previous variants is likely to reduce. Although no novel mutant has been discovered, the general trend is that the virus is becoming less severe, mainly because of enhanced immunity.Although it is believed that the omicron variant may likely not be the last mutant, it is expected that its effect will decrease with increasing immunity among the populace due to vaccines and infections. A previous study indicated that Omicron infection of vaccinated individuals, but not unvaccinated individuals, increases neutralizing activity against the Delta variant,145 Therefore, it is beneficial to administer a fourth dose of the vaccine to certain groups such as the elderly and immunocompromised individuals.147 Nevertheless, it is more crucial to complete the three doses of vaccination in a larger population than the fourth dose in healthy individuals, although we may need more boosters to maintain antibody levels in the long run. Furthermore, social distancing restrictions should not be lifted prematurely, as this can lead to unpredictable consequences.Furthermore, to effectively combat the Omicron variant and the pandemic in general, it is important to emphasize equal distribution of vaccines, especially in underdeveloped and developing regions. Presently, the three-injection vaccine can provide effective protection against Omicron, although there will be a percentage of breakthrough infections with milder symptoms. Some countries have approved the implementation of the fourth-injection vaccines. Recent researches show that the protection provided by the third dose of vaccines wanes over time, while the antibody levels can be restored by a fourth dose.


Moringa oleifera, which is extensively known for its nutritional properties, has been investigated in this work by preparation, characterization, and evaluation of the antioxidant , antifungal, photoprotective, and cytotoxicity profile against human melanoma Colo38 cell line of two different extracts  and one infusion of dry leaves collected from Paraguay in four distinct harvest times . The outcomes of this study highlight Moringa oleifera as a potential ally to counteract skin aging and oxidative stress, as indicated by the favorable antioxidant profile of the extracts and infusions of Paraguay, which was, in all cases, superior to that provided by the same plant species when collected from Senegal. Moreover, some samples were more efficient in preventing the photodegradation of UVA filter butyl methoxydibenzoylmethane (Avobenzone) compared to commercial filters, thus suggesting an interesting future role as natural additives in sunscreens.This research investigated plant extracts as a source of potential new actives in the nutritional, cosmetic, and pharmaceutical fields.  The natural environment provides a large variety of compounds suitable for medicinal use. Humankind has always searched for remedies in the vegetal world with the aim of maintaining or attaining a wellness state, at first by developing medical practices linked to the traditional and religious culture of every society and population, and later, with the advent of modern medicine, by searching for phytocomplexes and active compounds, finding inspiration for the development of drugs for various kinds of diseases.Products of natural origin will become increasingly important in the future of the cosmetic and pharmaceutical industries, in line with rising conscious and eco-friendly choices concerning, among other things, sunscreens, UV filters, and, more generally, photoprotection. In fact, there are many natural compounds endowed with photoprotection capabilities, for example through direct UV filtration, or synergism with other filters, or by counteracting side effects of commercial UV filter such as photodegradation or photosensitization .Antioxidant compounds such as flavonoids and polyphenols play a crucial role in preventing and/or quenching UV radiation-related ROS generation and could be considered as valid options to improve sun protection and to slow down photoinduced skin ageing . Some anMoringa oleifera is a medicinal plant used in various traditional medical practices and widely described in the scientific literature. It belongs to the genus of the Moringaceae family, which comprises 13 species found in sub-continental India, Kenya, and north and north-east Africa. The species Moringa oleifera is original to the sub-Himalayan region of northern India, Bangladesh, Pakistan, and Afghanistan, but is now widespread and cultured for health and food purposes in the tropical and subtropical areas. The favorable growing conditions for Moringa oleifera are 25–35 °C, at an altitude of 500 m under direct sunlight, and in slightly acid or alkaline soil (pH 5–9). However, it can tolerate different environmental conditions such as high temperatures up to 48 °C, low temperatures, altitude, and a broad variety of soil types. It thrives best under a tropical insular climate, it grows well in the humid tropics or hot dry lands, and can survive pauper soils and drought [It is called the “tree of life” or “miraculous plant” in several traditional medicines, as each fraction, including leaves and roots, is edible and rich in substances with valuable health and nutritional properties .Due to its beneficial effects, it constitutes a major part of countless traditional ethnomedical practices, and the main uses reported concern inflammatory states, fertility problems, and lifestyle-related diseases ,9,10,11.Moringa oleifera, including hypocholesterolemia, hypoglycemia, antimicrobic, anti-proliferative, antispasmodic, anti-inflammatory, and antioxidant activities [More recently, there has been a substantial increase in the number of scientific articles reporting various potential applications of various portions of tivities ,13.Moringa oleifera is a precious source of polyphenols, and its phenolic and flavonoid fractions have been extensively identified and characterized [cterized ,15,16.Moringa oleifera leaves were able to act as natural preservatives for food storage, being more effective in terms of both antioxidant and antimicrobial capacity than synthetic preservatives such as BHT (butylated hydroxytoluene) [Those natural antioxidant compounds are directly linked with the antioxidant activity of Moringa extract. Following the growing interest in the usage of natural antioxidants in commercial food supplements, recently, it has been reported that extracts of toluene) ,18.Dry leaves are rich in phenolic acids and flavonoids, such as quercetin and its disaccharide derivative rutin, along with chlorogenic acid and ferulic acid .Those compounds have also raised interest for their potential application as adjuvants for topical formulation, due to their side activities supporting the efficacy of sunscreen ,21,22.Overexposure to UV rays is the primary cause of sunburn, oxidative stress, and consequent degenerative processes such as photo-aging, inflammation, and skin-related diseases, including erythema, hyperpigmentation, or skin cancer ,24.In particular, UVB radiation, due to the shorter wavelength and higher energy, is mainly responsible for UVB-induced sunburn or erythema; it is also highly carcinogenic as it is responsible for direct interaction with biological macromolecules and for DNA/RNA photoinduced damage . UVA radFor these reasons, the antioxidant and antiproliferative activities, while exploring the multifunctionality profile of a new drug or natural extract, occupy a prominent position among the supplementary biological activities of a UV filter molecule or raw material ,25,26.Substantial skin photoprotective effects  are attributed to many polyphenolic natural compounds, due to their structural features such as hydroxyl and aromatic moieties that generally increase the UV absorption capacity .Topical application of those bioactive ingredients showed a decrease in UVB-induced skin sunburn events, suggesting and enforcing a definitive protective role for the skin from UV-related negative side effects .The raising of scientific literature regarding the photoprotective potential of natural compounds, however, has not led to the approval of any natural sunscreen. The combination of commercial UV filters (organic or inorganic) and vegetal extracts, instead, is stating itself as an affirmed formulation trend, due to interesting reports of increased UV absorption, booster activity, and/or prevention of UV-related damage ,29.Moringa oleifera extract for their potential in the cosmetic/medicinal field, focusing the characterization on natural compounds that could play a significant role in photoprotection. Moreover, sun-related skin damages are emerging as a complex pattern of mechanisms that would benefit from multifunctional molecules. Thus, to complete the characterization of a multifunctional profile of these extracts, in view of a possible topical application, the antiproliferative activity on the human melanoma cancer cell line Colo38 was investigated as well in order to possibly identify chemopreventive properties. This further investigation is supported by several examples of the antiproliferative effects of extracts of different fraction of M. oleifera reported in the literature [M. oleifera from Senegal, which outlined an interesting activity of the hydroalcoholic extract in the low micromolar range [Taking this into account, the rationale of this work was to characterize and biologically evaluate three types of terature ,31,32, aar range .Moringa oleifera extracts have been recently investigated against another important skin problem associated with the proliferation of fungi species [Among the biological activities,  species ,34,35. B® 530, Life Science UV/VIS spectrophotometer, Single Cell Module . The instrument used to conduct ORAC analysis was a Fluoroskan FL® ascent  with fluorescent filters , linked to Ascent Software ® (version 2.6) for data control and processing. In the sample loading phase, 96-well plates with a black background were used. PCL analyses were performed with a Photochem® apparatus  with a Double Bore ® phosphorus lamp . The spectrophotometric analyses were conducted with a UV–Vis spectrophotometer SHIMADZU UV-2600 240 V. Photostability studies were performed with Atlas Suntest CPS+ solar simulator, . WW5 PMMA plates were purchased from Schonberg GmbH . Microorganisms used in this study were Epidermophyton floccosum var. floccosum (The Netherlands), CBS 358.93 strain; Trichophyton tonsurans (The Netherlands), CBS 483.76 strain; Trichophyton mentagrophytes (The Netherlands), CBS 160.66 strain; Microsporum canis (Iran), CBS 131110 strain; Microsporum gypseum (Iran), CBS 130948 strain; Arthroderma cajetani (The Netherlands), CBS 495.70 strain; and Candida albicans (ATCC 10231). All dermatophytes were maintained at 4 °C as agar slants on SDA. Sabouraud dextrose agar (SDA) was purchased from Sigma-Aldrich SRL, Milano, Italy. HPLC analysis was performed using an Agilent 1100 Series HPLC System equipped with a G1315A DAD and with a Hydro RP18 Sinergi 80A column  from Phenomenex.All reagents and solvents were commercially available and used without further purification. The spectrophotometer used for antioxidant analysis was a Beckman Coulter™, DUMoringa oleifera leaves were collected in 2017 in February (lot number: FEB2017B1), March (lot number: MARZO2017B1), April (lot number: ABRIL2017B2), and May (lot number: MAYO2017B3) at Yhaca-Guazu . After collecting, the leaves were dried by MANITS AGROPY S.A. . Dried samples were packaged and sent to EDYNEA Srl , who supplied them to our laboratory. Before use, dried leaves were ground with a mortar until a fine powder was obtained, then stored at room temperature.The extraction processes were conducted by making some changes to the protocols previously defined by Vongsak et al.  and NoumHydroalcoholic extract: 10 g of dried leaf powder was placed for 1 h under magnetic stirring with 200 mL of hydroalcoholic solution  at room temperature. The residue was then filtered and concentrated in vacuo to provide the desired hydroalcoholic dry extract.Methanolic extract: The methanol dry extract was obtained by mixing about 5 g of powder with 100 mL of methanol and subjecting the mixture to 2 sonication cycles each of 40 min at 80%, followed by 9 min of centrifugation at 3500 rpm. The supernatant of the two cycles was pooled and concentrated in vacuo to obtain the desired dry methanol extract.Infusion: The infusion was obtained by adding 10 g of dried leaf powder to 150 mL of distilled water previously brought to boiling conditions, and then left in infusion for 30 min. The infused solution was then filtered and lyophilized to obtain the desired dried infusion.M. oleifera were tested to quantify the scavenging activity of DPPH radicals according to a procedure described by Wang et al. [The different extracts of g et al. . This in50 values were then calculated, defined as the amount of antioxidant needed to decrease the initial DPPH concentration by 50%. A total of 0.750 mL of extract solution at different concentrations was added to a DPPH methanolic solution (1.5 mL), and the absorbance was measured by a UV–Vis spectrophotometer. The IC50 values were determined by regression analysis of the data obtained at different sample concentrations and expressed as µg/mL and s.The IC3+) into ferrous ions (Fe2+) in the presence of TPTZ  in an acidic environment. The ferric-tripyridyl-triazine complex (Fe(III)-TPTZ) is reduced to the ferrous form (Fe(II)) in the presence of an antioxidant, and this reduction corresponds to an intense blue color with a maximum absorption peak at 593 nm. The FRAP reagent is a mixture of 0.1 M pH 3.6 acetate buffer, 10 mmol/L TPTZ in 40 mmol/HCl and 20 mmol/L ferric chloride in a 10/1/1 ratio and is prepared immediately before analysis. A total of 1.9 mL of FRAP reagent was added to 0.1 mL of diluted sample (or solvent when running blank). All samples were incubated in the dark at 37 °C for 10 min, and then the absorbance was measured at 593 nm with a UV–Vis spectrophotometer. A calibration curve was prepared using Trolox as a standard and the results were expressed as µmol of Trolox equivalent (TE) per gram of dry matter.The activity of the extracts was evaluated following the FRAP method by Benzie and Strain , which eThe ORAC test was performed on the basis of the previously modified Hong procedure . In this® phosphorous lamp, output 351 nm, 3 mWatt/cm2). The antioxidant capacity was measured using the ACL kit (Antioxidant Capacity of the Liposoluble substance) of the manufacturer. The kinetic light emission curve was monitored for 180 s and the final result was expressed as micromoles of Trolox (standard) per gram of dry sample. The areas under the curves were calculated using PCL soft control and analysis software.The PCL test is able to measure the antioxidant capacity of a sample against superoxide anion radicals. It is based on the method of Popov and Lewin  and quanM. oleifera were solubilized in methanol and suitably diluted before the analysis, while the formulations were suitably treated to extract the component to be tested. The antioxidant test was carried out in triplicate for each sample.The extracts of M. oleifera extracts [An adapted and optimized Folin–Ciocalteu method was used to evaluate the total polyphenol content in extracts . Gallic 3PO4) as solvent A and acetonitrile (0.01 M H3PO4) as solvent B, were programmed to gradient, using the following ratios: 90:10 (A/B) to 80:20 (A/B) in 5 min, maintained for 5 min, 80:20 (A/B) to 20:80 (A/B) in 10 min, 20:80 (A/B) to 90:10 (A/B) in 2 min, with a flow rate of 1.2 mL/min at room temperature. The injection volume for all samples and standards was 5 µL. Standard solutions of nicotinic acid, ellagic acid, rutin, chlorogenic acid, ferulic acid, and quercetin were prepared in methanol and properly diluted with methanol to obtain final concentrations between 80 and 0.625 µg/mL. An accurately weighed aliquot of each extract was dissolved in methanol or water–methanol (50:50). Before injection, each solution was filtered through a 0.45 μm nylon membrane filter and analyzed in triplicate by HPLC. The quantitative HPLC analysis was calculated, for each compound, according to its peak area.HPLC analysis was performed using an Agilent 1100 Series HPLC System equipped as describe above. The separation was monitored at an absorbance value of 254 ± 8 nm. The elution conditions, using water :The in vitro antifungal activity against the six selected dermatophytes was evaluated using the previously described plaque growth inhibition method . Each exCandida albicans suspension was prepared in an aliquot of 5 mL of sterilized water. Stock solutions were prepared by dissolving the extracts and fluconazole in DMSO at a concentration of 12.80 mg/mL. Minimal inhibitory concentration (MIC) of the extracts was performed by the broth microdilution method according to the Clinical and Laboratory Standards Institute/National Committee for Clinical Laboratory Standards (CLSI/NCCLS) Approved Standard M27-A3, 2008 (NCCLS) as previously described [escribed .Moringa oleifera samples  were added under stirring. In the case of the formulations containing Avobenzone, this was incorporated in the oil phase when the temperature was equal to 40 °C. The final pH of the formulations was brought to 6 with 10% NaOH.A basic topical O/W formulation was prepared. The components of the hydrophilic phase were weighed and added in succession in a beaker as follows. Water and glycerin were brought to 60 °C. Xanthan gum and phenoxyethanol and ethylhexylglycerin were then added via a turbo emulsifier. The components of the oil phase were weighed separately and brought to a temperature of 70 °C, until solubilization of the ingredients. After that, the oil phase was slowly added to the aqueous phase under constant stirring with an ULTRA-TURRAX turbo emulsifier. Finally, INCI: Aqua, glycerin, xanthan gum, phenoxyethanol (and) ethylhexylglycerin, potassium olivoyl hydrolysed wheat protein, cetearyl alcohol, cocoglycerydes, coco-caprylate.2 PMMA plates. The plates were weighed before and after the spread. Three plates were prepared for each product and five measurements were made of each plate. Before taking the measurement, the plates were located away from light, at room temperature, for 15–30 min. Finally, UV transmittance measurements from 290 to 400 nm were performed using a UV–Vis spectrophotometer. The blank was prepared using a plate coated with 15 μL of glycerin due to its non-fluorescent and UV transparency. The in vitro SPF, UVA-PF, and critical wavelength parameters were performed using the SPF Calculator Software , Shimadzu, Milan, Italy) as previously described [A precisely weighed amount of 0.0320 ± 0.0005 g of each formulation was spread in circular motions on 25 cmescribed .2. Before and after the exposure, the spectral transmittance of thin film of sunscreen was recorded from 290 to 400 nm. According to Equations (3) and (4), the residual percentages of SPF in vitro (% SPFeff.) and UVA-PF (% UVA-PFeff.) were calculated, respectively.Each selected extract, alone or in combination with commercial UV filters, was incorporated in an oil-in-water (O/W) emulsion and spread on a PMMA plate. PMMA plate was irradiated with a solar simulator applying different UVA dose equivalents to an effective erythema radiant exposure of 150 kJ/mMoringa oleifera leaves) were studied on two different human cell lines: the melanoma Colo 38 cells and the non-tumoral keratinocyte HaCat cells [The potential antiproliferative effects of the most interesting samples  and incubated for another two days. After 24 and 48 h, the cell suspensions were mixed and counted with a Z2 Coulter Counter .The melanoma Colo 38 cells were cultured in RPMI 1640 medium , with 10% fetal bovine serum (FBS)  and 100 U/mL penicillin/streptomycin ; the pH of the medium was 7.2 and all the cell cultures were incubated at 37 °C in a 5% CO2 atmosphere. A total of 25,000 cells/mL were seeded in 24-well plates in RPMI complete medium. Eight hours after seeding, the compounds were added in serial dilutions to obtain different concentrations , and incubated for another 24 h. Then, cells were detached, resuspended in fresh complete medium, and counted with a Z2 Coulter Counter .The keratinocyte HaCat cells were maintained in DMEM (4.5 g/L glucose)  with 10% FBS , 100 U/mL penicillin/streptomycin , and 2 mM glutamine ; the medium pH was 7.2 and all the cultures were incubated at 37 °C in a 5% COp ≤ 0.05) were given where appropriate for all data collected. One-way ANOVA and Least Significant Difference (LSD) post hoc Tukey’s honest significant difference test were used for comparing the bioactive effects of different samples. Statistical software STATISTICA 6.0  was used for all computations.Relative standard deviations and statistical significance  were compared in order to evaluate the variation in the activity profile, both in qualitative and quantitative terms. From each sample, two extracts  and an infusion were prepared for a total of 12 lyophilized samples, which were then investigated for their potential antioxidant, antifungal, UV-filtering, and photostabilizing activity. Different methods are available to measure the antioxidant capacity of natural extracts, whose components can be more or less specific towards the distinct radical species. In this work, we decided to evaluate the antioxidant activity of the extracts and infusions by means of DPPH, FRAP, ORAC, and PCL tests to obtain a broad in vitro activity profile  [The results obtained from the FRAP test are in line with those of the DPPH test. Again, the highest values were returned by the three extracts of February together with the hydroalcoholic extract of March. The lowest value obtained came from the infusion of March (384.56 ± 9.20 µmol TE/g), although it is superior when compared to the infusion of the leaves of M. oleifera from Senegal (2942.8 ± 27.28 µmol TE/g) [The ORAC values showed a suitable activity profile of all the extracts with similar values. The hydroalcoholic extract of February, even in this case, had the best profile, with over twice the antioxidant activity of the extract of dried leaves of ol TE/g) .M. oleifera from Senegal. The lowest value relative to the antioxidant profile of M. oleifera of Paraguay was shown by the hydroalcoholic extract of the month of May (397.5 ± 14.14 µmol TE/g), which has still a better profile when compared with the lowest value obtained from the same plant from Senegal .PCL data confirmed February extracts as the best antioxidant candidates, with antioxidant capacity values of 689.24 and 640.64 µmol TE/g for hydroalcoholic and methanolic extract, respectively. The results obtained from the extracts of the months of March, April, and May presented a strong profile as well. The hydroalcoholic extract of March, especially, showed an activity comparable, albeit slightly higher, to that previously reported for the infusion (512.1 ± 10.30 µmol TE/g) and hydroalcoholic extract (506.8 ± 3.19 µmol TE/g) of leaves of The quantitative determination of the total content of polyphenols in the 12 extracts provided the data presented in M. oleifera leaves was found to be considerable—indeed, all the extracts showed a clearly better profile with respect to our previously reported data [The total polyphenol content of the extracts of ted data .M. oleifera from Paraguay.The hydroalcoholic extracts of February and March, followed by the methanolic extract of February, displayed the highest content of polyphenols, equal to 82.51, 75.16, and 73.64 µmol GAE/mg, respectively. Those results are in agreement with the antioxidant tests we carried out: the greater the total polyphenolic fraction detected, the greater the antioxidant capacity. The above results of the Folin–Ciocalteu test highlighted the presence of a polyphenolic component in the extracts and infusions of leaves of the four months of harvesting of Characterization of the aforementioned extracts and infusion was performed through HPLC analysis, in order to identify and quantify compounds of interest. Our attention focused on phenols known for playing an active role in the field of photoprotection ,21,22: cThe quantitative estimation of the selected actives, according to peak area, is given in Moringa oleifera. The hydroalcoholic extracts of February, March, and April were rich more than ferulic acid, while the infusions showed higher contents of chlorogenic acid, which indeed was the active with the highest percentage in the infusion of May. It should be noted that quercetin is present only in the hydroalcoholic and methanolic extracts of the month of May. This aspect highlights the different interactions between the phytohormones of the plant, light, water availability, and temperatures that may occur at various moments of the year. While no significant quantitative variation was noted among the four harvest times of M. oleifera from Paraguay, we found some interesting differences with the quantitative composition of M. oleifera leaf extracts from Senegal [The results summarized in  Senegal , where r Senegal .Microsporum canis, Microsporum gypseum, Trichophyton tonsurans, Epidermophyton floccosum, Trichophyton mentagrophytes, and Arthroderma cajetani) with the diffusion method in sabouraud dextrose agar (SDA). The results (data not shown) indicated E. floccosum as the mushroom that is most sensitive to the inhibitory activity of Moringa oleifera extracts, with discrete results obtained by the extracts of the months of February  and of March, except for the infusion, with values of 31.65% and 21.52% for the hydroalcoholic and methanolic extract, respectively. All the other data referred to a weak activity; for some dermatophytes, the treatment with the vegetal extract seems to have slightly stimulated, rather than inhibited, the fungus growth.An antifungal is essential to fight skin infections, and the excessive production of radicals is the body’s self-defense mechanism. In this regard, many secondary plant metabolites have been used as actives against fungal infections caused by dermatophytes . It was Candida Albicans was evaluated, and MIC (minimum inhibitory concentration) was calculated in order to establish the potency of the samples. However, neither of them showed any activity against Candida Albicans in the range of 0.0244–12.48 mg/mL after 24 and 48 h (data not shown).Furthermore, a possible inhibitory effect against After we established the antioxidant capacity of the raw extracts, being topical application the final aim of those materials, we carried out the PCL test on twelve O/W formulations containing 5% of each extract or infusion from the four harvest times considered in this work, in order to establish if the strong antioxidant parameters obtained from the extract were maintained once incorporated into a cosmetic product. The results are reported in As reported, the antioxidant capacity of the extracts is retained when they are formulated in a cosmetic vehicle, with values that are in line with those reported for the raw extracts, taking final concentration into account. No substantial variation, as observed for the raw extracts, was detected among the four harvest periods, with the hydroalcoholic extract from February leading the ranking in terms of antioxidant performance.The in vitro SPF determination was performed in order to evaluate the filtering profile of the extracts when included in suitable cosmetic formulations compatible with topical application. An O/W green emulsion was chosen as vehicle, and the hydroalcoholic and methanolic extracts and the infusion were incorporated at 5%. All tested samples returned SPF values among 2 (data not shown), which could be considered valuable in the context of vegetal extracts: this value means that 50% of UVB radiation is filtered by the finished product, and phytocomplexes are usually heterogeneous and contribute to sun ray protection through other complementary mechanisms .Recently, some ingredients of sunscreens have emerged as potential boosters, being able to improve the total SPF value of some filters, also acting as photostabilizers, contrasting the degradation of unstable UV filters through quenching of the reactive radical species generated after UV irradiation .Moringa oleifera extracts emerging for the above-mentioned studies  towards the commercial UVA filter butyl methoxydibenzoylmethane (Avobenzone). This organic filter, although very active and inexpensive, is known to be photo-unstable due to keto-enolic tautomerism induced by UV irradiation, and must be used in combination with other UV filters that act as stabilizers, such as Octocrylene. These filters have both encountered several concerns in their application, being banned in the so-called “tropical paradise” of Haway and Palau for their negative impact on the marine environment and also for their skin irritation problems due to photosensitization. In the perspective of a “green” alternative, natural antioxidants may be promising options to improve Avobenzone photostability [In this context, we decide to explore the potential photostabilizing effect of the best tability . The twotability , we deciTo evaluate the efficiency of the extracts on the stability of Avobenzone, the formulations were subjected to SPF analysis before and after UV irradiation. According to regulatory criteria, if a filter shows a residual percentage of SPF in vitro and UVA-PF greater than or equal to 80, after irradiation, then it can be considered photostable . The resAs expected, Avobenzone showed a marked photodegradation after UV exposure, with UVAPF and SPF values decreased by 22.33% and 16.23% respectively. Those data confirm its photo-unstable behavior in terms of UVA protection. The combination with Octocrylene (5%) furnished better post-irradiation filtering values, with 9.37% and 9.13% degradation of UVAPF and SPF values, respectively.The two extracts and the infusion alone showed nearly quantitative photostability, with the slight exception of the methanolic extract, which showed degradation values below 5%.The combination of the two extracts and the infusion with Avobenzone brought us some outstanding results: in all cases, the vegetal extract was able not only to completely avoid photodegradation, but in some way to improve the filtering parameters after irradiation . Even if it is not possible to define this behavior as a “booster” effect, those data collectively outline the potentialities of multifunctional herbal extracts as synergic agents with UV filters, and their ability to potentiate the photoprotection in terms of both SPF and UVAPF.Finally, the combination of ferulic acid at the given concentrations and Avobenzone result in a slight decrease in the filtering parameters , corroborating the hypothesis that the stabilizing effect is due to a synergic effect of all the distinct components of the extract. Nevertheless, we found that ferulic acid was still able to inhibit photodegradation even at very low concentrations, if compared to the commercial filter Octocrylene—this confirms its ability to act as “enhancer” of anti-UVB and anti-UVA protection .Moringa oleifera leaf extracts and the infusion from February, which showed strong antioxidant activity and photostabilization capacity, were tested on the human melanoma Colo38 cell line and immortalized human keratinocytes HaCat cell line. Cells were seeded and cultured in the presence of increasing concentrations of M. oleifera extracts . Then, after 24–48 h of treatment, cells were harvested and analyzed with a Beckman Coulter Counter. IC50 was calculated when cells were in the growth logarithmic phase, and indicates the drug concentration required for 50% cell growth inhibition. These preliminary results, summarized in 50 > 230.32 ± 18.90 µg/mL), also using the higher concentration of all the tested extracts on the Colo38 cell line and with the infusion treatment (IC50 > 500 µg/mL) on the HaCat cells.With the aim to exclude possible side effects, Moringa oleifera from Paraguay, comparing the activity profile of four different harvest times  through three different extraction techniques  for each month, with a total of 12 samples tested. Overall, all the tested extracts were superior in terms of antioxidant capacity and total polyphenol content when compared to the same plant from Senegal [M. oleifera from Senegal, was detected in trace amounts only in the extracts of May, an aspect that highlights the variability in the production of secondary metabolites by the plant depending on several aspects that contribute to its growth . The 12 extracts were tested for possible inhibitory growth activity towards six different dermatophytes and Candida albicans, but no inhibitory activity worth mentioning was obtained. Concerning the potential photoprotective activity and photostabilizing capacity of the extracts, values obtained from the SPF analysis of all the extracts tested were among 2, considered to be of interest due to other possible photoprotective mechanisms exerted by the vegetal extract. As proof of this, the combination of the extracts and the infusion from February and the organic UVA filter Avobenzone proved to increase the photostability of Avobenzone itself more efficiently than the commercial filter Octocrylene, returning UVAPF and SPF values after irradiation even higher than before irradiation. This is not to be considered a booster effect, but it points out the potential of extracts of Moringa oleifera leaves as solar formulation additives, both for their antioxidant and photostabilizing activity.Following the most recent trends in sunscreen formulation and UV protection and the increasing attention given to natural products able to play a part in skin photo-aging and oxidative stress ,53, in t Senegal , with thMoringa oleifera leaf extracts from Paraguay can be considered as potential natural alternatives for sunscreen formulations, as proved by a set of biological activities, antioxidants and photostabilizers above all, that can synergistically improve skin photoprotection and reduce skin-related damages derived from excessive UV exposure. Considering the preliminary data on the antiproliferative effects on the HaCat cell line showed by the two extracts, the infusion from February emerges as the best candidate. Further studies will be conducted to establish the photostabilizing ability of these extracts even at lower concentrations than those considered in this work.To summarize,


The regular practice of physical activity helps in the prevention and control of several non-communicable diseases. However, evidence on the role of physical activity in mitigating worsening clinical outcomes in people with COVID-19 is still unclear. The aim of this study was to verify whether different levels of physical activity provide protection for clinical outcomes caused by SARS-CoV-2 infection. A cross-sectional study was conducted with 509 adults  with a positive diagnosis of COVID-19 residing in Ribeirão Preto, São Paulo, Brazil. Participants were interviewed by telephone to determine the severity of the infection and the physical activity performed. Binary logistic regression was used to indicate the odds ratio (OR) of active people reporting less harmful clinical outcomes from COVID-19. Active people had a lower chance of hospitalization, fewer hospitalization days, less respiratory difficulty and needed less oxygen support. The results suggest that active people, compared to sedentary people, have a lower frequency of hospitalization, length of stay, breathing difficulty and need for oxygen support. These results corroborate the importance of public policies to promote the practice of physical activity, in order to mitigate the severity of the clinical outcomes of COVID-19. The Severe Acute Respiratory Syndrome of the Novel Coronavirus SARS-CoV-2) has infected more than 525 million people and caused more than 6 million deaths . Among t has infeAmong the risk factors mentioned above, the scientific evidence linking a sedentary lifestyle with chronic diseases and premature death is very well established. In contrast, being physically active is one of the main determinants of health , as it rRecognizing the known positive effects of physical activity on immunity, inflammation and infection ,25, we hA cross-sectional observational study was performed; the data collection process took place between 7 June and 27 December 2020. This manuscript followed the guidelines from the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) .® version 3.043  was adopted for sample size calculation.The sample consisted of adults of both sexes, who had a positive diagnosis of COVID-19. Inclusion criteria were: people ≥ 18 years of age, positive diagnosis for COVID-19, living in Ribeirão Preto, state of São Paulo—Brazil. Exclusion criteria were: presence of any conditions of immunological compromise, prolonged use including the use of corticosteroids and/or chemotherapy, transplant patients, presence of neurodegenerative diseases. The sample size calculation considered a prevalence of hospitalizations for COVID-19 of 10.0%, a precision of 3.5% and a confidence interval of 95%, for a finite population of 33,643 infected during data collection, achieving a sample minimum of 500 to 650 participants. The Power and Sample Size Program®, seven evaluators, trained and supervised by the principal investigator, made 3814 telephone calls. Of this total, after a maximum of three attempts, 647 participants answered the calls. Data from a total of 14 individuals were disregarded, as the participants did not meet the eligibility criteria, and 124 persons did not wish to participate in the study. Thus, 509 people diagnosed with COVID-19 were interviewed and considered eligible  (CAAE n. 39645220.6.0000.5393), following the guidelines that regulate research involving human beings according to the Resolution of the National Health Council (CNS) 466/12. Subsequently, the project was sent for consideration by the Municipal Health Department of Ribeirão Preto and approved . This entity provided personal information  of people diagnosed with COVID-19. The names were randomized to avoid the risk of bias in the study. Participants were contacted by telephone and invited to participate in the study by answering the questionnaire “Profile of the person diagnosed with COVID-19” and the short version of the International Physical Activity Questionnaire (IPAQ). The free Google FormsInitially, the researchers designed a pilot version of the questionnaire based on the research goals of the study. Next, the questionnaire was sent to a committee of judges, consisting of three Ph.D. professionals, such as a Kinesiologist, a Nurse, and a Physician, who were familiar with the research objectives of the study. For each question, the specialist responded “I totally disagree”, or “I disagree” or “Indifferent/neutral”, or “I agree” or “I totally agree”. In addition, it was necessary to answer the question “Do you suggest any modification to this question?” Each answer was analyzed by the study researchers, and when two or more experts marked the same alternative, it was accepted by the researchers. The suggestions for some modifications to the question were also taken into account. This approach is consistent with recommended approaches to the establishment of the content validity of questionnaire surveys ,30,31,32The questionnaire had four pages and was grouped into the following parts: Identification , Block 1 , Block 2 (contains 17 questions that refer to the period before the diagnosis by COVID-19), Block 3 , Block 4 , Block 5  .The questionnaire included questions regarding personal information such as weight and height ), age, sex, profession, family income, education level, marital status, smoking and drinking habits and pre-existing diseases. In addition, addressed the need for hospitalization, how many days a patient remained hospitalized, respiratory difficulties ) , oxygen Physical activity was measured using the Brazilian-validated short version of the International Physical Activity Questionnaire (IPAQ-SV) ,33. The ® and validated using double key data entry and verification. This procedure was used to ensure the highest accuracy and quality of the data collected. The variables sex, range age, family income, education level, BMI and clinical outcomes of COVID-19 were presented by absolute (n) and relative (%) frequency. Clinical outcomes examined dichotomously were: hospitalization, hospitalization days, breathing difficult, oxygen support, intubation, days of intubation and death. Fisher’s exact test was used to verify the association between sedentary/physically active people and clinical outcomes in people diagnosed with COVID-19. Binary logistic regression was adopted to indicate the odds ratio (OR) of the physically active group reporting mitigated clinical outcomes of COVID-19 when adjusted for confounding variables including, age, family income, education level and BMI. All analyses were performed using SPSS version 20 , significance level was set to α ≤ 5%.Data were entered in Microsoft ExcelA total of 509 individuals participated in the study (women = 61%). Of these, 49.3% were classified as sedentary. The age range was from 18 to 89, with 78% being between 18 and 59 years old and 22% being 60 years old or older. Regarding socioeconomic status and education level, 87% had a family income above BRL 908.00 and 69% had completed high school, respectively. Regarding nutritional status, 72% were overweight or obese. Related to clinical outcomes, 9% required hospitalization, 6.5% were hospitalized for 6 days or more, 7.9% had breathing difficulty, 7.9% required oxygen support, 2% required intubation, 1.8% were intubated for 6 days or more and 1% died .When we performed Fisher’s exact test, no statistically significant association was found among people aged 18 to 59 years, sedentary and active for all clinical outcomes. However, a statistical significant association was found among people aged ≥60 years old, sedentary and active, for the following clinical outcomes: hospitalization, breathing difficulty and oxygen support .When we performed Fisher’s exact test considering the whole sample (without grouping by age) statistically significant association was found between sedentary and active people for the following clinical outcomes: hospitalization, hospitalization days, breathing difficulty and oxygen support .p = 0.017), hospitalization days , breathing difficulty  and oxygen support . The same did not occur for the clinical outcomes intubation , days of intubation  and death   .The study sought to verify whether physical activity levels are associated with the clinical outcomes of people diagnosed with COVID-19. The results, without age group classification, indicate that a physically active lifestyle has beneficial effects on the clinical outcomes of hospitalization, hospitalization days, breathing difficulty and oxygen support. However, considering the age group between 18 and 59 years, no difference was found between sedentary and active people. For the age group 60 years and over, an association was found between sedentary and active for the clinical outcomes of hospitalization, breathing difficulty and oxygen support. To the best of our knowledge, this is the first study to examine the relationship between meeting or not meeting physical activity recommendations (active versus sedentary) with different COVID-19 clinical outcomes. The relationships between self-reported physical activity and the different outcomes of COVID-19 studied are as follows:Our results show that physically active people had a lower risk of hospitalization when compared to sedentary people. Other studies have also reported similar results. Sallis et al.  through The lower frequency of hospitalization observed among the physically active subjects in the present study can be explained by the protective effects conferred by the regular practice of physical activity. For example, reduction of comorbidities and stress , and impIn our study, physically active participants had a shorter hospital stay when compared to sedentary participants. This finding is consistent with the results of a study carried out in South Korea . This asRegarding the need for oxygen support, the associations observed in the present study showed that physically active participants need less oxygen support compared to sedentary people. This lower need for oxygen support was also observed in the study by Mistry and Natesan, when they compared sedentary individuals with another who performed moderate-intensity physical activity . In our Regarding the need for oxygen support, the associations observed in the present study showed that physically active participants needed oxygen support less frequently than sedentary participants. This was also observed in a prior study that compared moderately active and sedentary groups .Physical activity can help improve immune system response and mitigate viral infection. In addition, being physically active prevents and treats numerous complications associated with COVID-19, such as heart disease, and neurological and metabolic disorders, including the positive effect on the renin–angiotensin system . ReducinIn our study, no difference was found between physically active and sedentary people regarding the need and duration of intubation. A study conducted in South Africa observed that people with moderate to vigorous levels of physical activity had a lower frequency of intubation compared with people with lower levels of physical activity . It shouRecent evidence points out that being physically active can reduce the mortality rates of COVID-19 . AlthougThe results found may change over time due to the continuity of the pandemic and the emergence of new variants.Despite the promising results obtained in this study, some limitations are present and must be considered. The observational and cross-sectional design of the study does not clearly allow for a cause-and-effect relationship  between Our investigation is one of the few to examine physical activity and clinical outcomes of COVID-19 in Brazil using telephone interviews. The present study was conducted prior to the initiation of the COVID-19 vaccination in Brazil, diminishing the risk of bias related to vaccination status. The International Physical Activity Questionnaire used in this study is easy to apply, has been widely used, has been validated in different populations and countries, including Brazil ,51, and Physically active people have a better clinical prognosis regarding the outcomes of COVID-19, especially with lower frequency of hospitalization, hospitalization days, respiratory difficulty and oxygen support. Our study provides evidence that active behavior is a modifiable risk factor for COVID-19 clinical outcomes. The global effort to encourage the population to adopt a physically active lifestyle, and avoid a sedentary lifestyle, may be a promising strategy for protecting and mitigating the severity of the clinical outcomes of COVID-19.


The increasing use of high-quality imaging techniques together with improved access to healthcare has led to an increase in the detection of thyroid nodules. Fine-needle aspiration cytology (FNAC) is currently considered the most accurate examination for the assessment of thyroid nodular disease. However, in about 25% of cases, FNAC leads to the diagnosis of an indeterminate thyroid nodule, which represents a problem because malignancy, although relatively low (up to 30%), cannot be excluded with certainty. According to the 2015 American Thyroid Association guidelines, patients with thyroid nodular disease with an indeterminate cytology can undergo, based on established factors, a total thyroidectomy or a hemithyroidectomy. However, if an intermediate or high-risk differentiated thyroid carcinoma is detected after the hemithyroidectomy, through histological examination, the above-mentioned guidelines recommend performing a completion thyroidectomy. The main aim of this study was to assess the rate of complications in patients undergoing a completion thyroidectomy after a hemithyroidectomy for a thyroid nodule with an indeterminate cytology.There is still controversy as to whether patients undergoing a completion thyroidectomy after a hemithyroidectomy for a thyroid nodule with an indeterminate cytology have a comparable, increased or decreased risk of complications compared to those submitted to primary thyroid surgery. The main aim of this study was to investigate this topic. Patients undergoing a thyroidectomy for thyroid nodular disease with an indeterminate cytology in four high-volume thyroid surgery centres in Italy, between January 2017 and December 2020, were retrospectively analysed. Based on the surgical procedure performed, four groups were identified: the TT Group , HT Group (hemithyroidectomy), CT Group (completion thyroidectomy) and HT + CT Group (hemithyroidectomy with subsequent completion thyroidectomy). A total of 751 patients were included. As for the initial surgery, 506 (67.38%) patients underwent a total thyroidectomy and 245 (32.62%) a hemithyroidectomy. Among all patients submitted to a hemithyroidectomy, 66 (26.94%) were subsequently submitted to a completion thyroidectomy. No statistically significant difference was found in terms of complications comparing both the TT Group with the HT + CT Group and the HT Group with the CT Group. The risk of complications in patients undergoing a completion thyroidectomy after a hemithyroidectomy for a thyroid nodule with an indeterminate cytology was comparable to that of patients submitted to primary thyroid surgery . The increasing use of high-quality imaging techniques together with improved access to healthcare has led to an increase in the detection of thyroid nodules, which have thus become a common clinical problem. At the same time, there has been an increase in the diagnosis of thyroid carcinoma, which has increased by 300% worldwide over the last three decades. The increase in the incidence rate of thyroid cancer is almost entirely due to the increase in the diagnosis of differentiated thyroid carcinoma (DTC), whose mortality has however remained relatively low and stable ,2,3,4.Fine-needle aspiration cytology (FNAC) is currently considered the most accurate examination for the assessment of thyroid nodular disease, given its high specificity 92%) and sensitivity 83%). However, in about 25% of cases, the FNAC leads to the diagnosis of an indeterminate thyroid nodule, which represents a problem because malignancy, although relatively low (up to 30%), cannot be excluded with certainty. In patients with this diagnosis, based on the risk of malignancy established through preoperative investigations, surgery or surveillance may be indicated. As regards surgery, in these patients, it has a diagnostic rather than therapeutic purpose, with almost 80% of surgical procedures unnecessary [3%. Howev2% and seAccording to the 2015 American Thyroid Association (ATA) guidelines, patients with thyroid nodular disease with an indeterminate cytology can undergo a total thyroidectomy or a hemithyroidectomy. The choice between these two surgical procedures is influenced by several factors, including: a nodule size greater than 4 cm, a familial history of DTC, previous radiation exposure, highly suspicious ultrasound features and positivity for known mutations specific for DTC. In addition, all these factors must be further associated with the presence of bilateral nodular disease, the possible coexistence of hyperthyroidism, the patient’s medical comorbidities and patient preference. However, if an intermediate or high-risk DTC is detected after a hemithyroidectomy, through final histological examination, the above-mentioned guidelines recommend performing a completion thyroidectomy, as a hemithyroidectomy is considered oncologically inadequate in these cases [There is still controversy as to whether patients undergoing a completion thyroidectomy for this reason have a comparable, increased or decreased risk of complications compared to those submitted to primary thyroid surgery ,20,21,22The main aim of this study was to assess the rate of complications  in patients undergoing a completion thyroidectomy after a hemithyroidectomy for a thyroid nodule with an indeterminate cytology.This is a multicentre, retrospective, observational study on patients who underwent thyroidectomy for thyroid nodular disease with indeterminate cytology between January 2017 and December 2020.-Multi-specialty General Surgery Unit, Cagliari University Hospital, Monserrato (CA);-General, Oncologic, Mininvasive and Bariatric Surgery Unit, University Hospital “L. Vanvitelli”, Naples (NA);-Academic General Surgery Unit “V. Bonomo”, Bari University Hospital, Bari (BA);-General Surgery Unit, Prato Hospital “Santo Stefano”, Prato (PO).Data were collected from four high-volume thyroid surgery centres in Italy:All participating centres used the same dedicated Microsoft Office Excel database  for data collection.Only patients undergoing conventional open thyroidectomy were included in this study. Patients simultaneously submitted to parathyroidectomy, those with preoperative diagnosis of lymph node metastasis, those with suspected medullary thyroid carcinoma  and those with incomplete data were excluded.Demographic and preoperative data, information about the surgical procedure, postoperative stay, histological findings and complications were assessed.-TT Group: total thyroidectomy;-HT Group: hemithyroidectomy ;-CT Group: completion thyroidectomy;-HT + CT Group: hemithyroidectomy with subsequent completion thyroidectomy .Based on the surgical procedure performed, four groups were identified:The primary endpoint was to assess the occurrence of complications  in patients undergoing completion thyroidectomy.-TT Group with HT + CT Group ;-HT Group with CT Group .For this purpose, we compared:The secondary endpoint was to compare HT Group with CT Group in terms of operative time.In all enrolled patients, preoperative evaluation included: medical history, physical examination, blood tests to assess thyroid function, high-resolution ultrasound (US) of the neck and fine-needle aspiration cytology.Hyperthyroidism status was defined in the case of low serum thyroid-stimulating hormone (TSH) (<0.4 mIU/L) and use of thyrostatic drugs.High-resolution US of the neck was always performed before surgery by an experienced surgeon, with careful evaluation of the thyroid gland and cervical lymph nodes.The results of fine-needle aspiration cytology were classified according to the 2014 Italian consensus of the Italian Thyroid Association (AIT), the Italian Association of Clinical Endocrinologists (AME), the Italian Society of Endocrinology (SIE) and the Italian Society for Anatomic Pathology and Cytology joint with the Italian Division of the International Academy of Pathology (SIAPEC-IAP) for the classification and reporting of thyroid cytology .Moreover, all patients underwent preoperative fibrolaryngoscopy to assess vocal cord mobility.The indication and type of initial surgery  were established according to the 2015 American Thyroid Association guidelines. After final histological examination, completion thyroidectomy, with or without central neck dissection, was performed in accordance with the same guidelines [Recurrent laryngeal nerves and parathyroid glands were systematically searched and identified.Intraoperative nerve monitoring (IONM), energy-based device and drain were used according to the preference of the operating surgeon.The duration of the surgical procedure was estimated, in minutes, from skin incision to skin closure.Postsurgical hypoparathyroidism was defined as iPTH < 10 pg/mL following the operation . Permanent hypoparathyroidism was defined as iPTH levels below the normal range for more than 12 months.Recurrent laryngeal nerve injury was diagnosed through postoperative fibrolaryngoscopy. After surgery, fibrolaryngoscopy was performed in the case of suspected recurrent laryngeal nerve injury for loss of signal at IONM or hoarseness. Recurrent laryngeal nerve injury was defined as permanent in the case of vocal cord paralysis, identified through fibrolaryngoscopy, persisting for more than 12 months.® 20.106. Chi-squared test or Fisher exact test were used for categorical variables and t-test or Mann–Whitney U test for continuous variables. p values < 0.05 were considered statistically significant.Statistical analyses were performed with MedCalcA total of 751 patients met the established inclusion criteria: 242 (32.22%) males and 509 (67.78%) females, with a mean age of 51.74 ± 14.15 years.As for the initial surgery, 506 (67.38%) underwent a total thyroidectomy (TT Group) and 245 (32.62%) a hemithyroidectomy (HT Group). Detailed data regarding features of the whole sample and of these two groups are reported in Among all patients submitted to a hemithyroidectomy, 66 (26.94%) subsequently underwent a completion thyroidectomy. In patients undergoing a hemithyroidectomy with a subsequent histological finding of malignancy, a completion thyroidectomy was performed in 56.90% of the cases. One (1.52%) patient underwent a two-stage thyroidectomy for a loss of signal at intraoperative nerve monitoring. A central neck dissection was performed in 13 (19.70%) patients. The average interval between an HT and a CT was 2.12 ± 1.20 months. Malignancy in the contralateral lobe was found in 16 (24.24%) patients, while lymph node metastasis was found in 9 (13.64%).These results are shown in In the TT Group, there were 141 (27.87%) cases of transient hypoparathyroidism, 12 (2.37%) cases of permanent hypoparathyroidism, 4 (0.79%) cervical haematomas, 17 (3.36%) unilateral recurrent laryngeal nerve lesions, 13 (2.57%) transient recurrent laryngeal nerve lesions, 4 (0.79%) permanent recurrent laryngeal nerve lesions and 1 (0.20%) wound infection. No bilateral recurrent laryngeal nerve injury was observed.In the HT + CT Group, there were 14 (21.21%) cases of transient hypoparathyroidism, 2 (3.03%) cases of permanent hypoparathyroidism, 4 (6.06%) unilateral recurrent laryngeal nerve lesions, 3 (4.55%) transient recurrent laryngeal nerve lesions and 1 (1.52%) permanent recurrent laryngeal nerve lesion. No bilateral recurrent laryngeal nerve injury, cervical haematoma or wound infection was observed.No statistically significant difference was found in terms of the occurrence of hypoparathyroidism, cervical haematoma, recurrent laryngeal nerve injury and wound infection between the two groups.These data are reported in In the HT Group, the median operative time was 50 min . Intraoperative nerve monitoring, an energy-based device and a drain were used in 140 (57.14%), 245 (100%) and 73 (29.80%) patients, respectively. The average postoperative stay was 1.72 ± 0.76 days. About complications, there was one (0.41%) cervical haematoma, seven (2.86%) unilateral recurrent laryngeal nerve lesions, five (2.04%) transient recurrent laryngeal nerve lesions and two (0.82%) permanent recurrent laryngeal nerve lesions. No wound infection was observed.In the CT Group, the median operative time was 50 min . Intraoperative nerve monitoring, an energy-based device and a drain were used in 33 (50%), 64 (96.97%) and 19 (28.79%) patients, respectively. The average postoperative stay was 1.77 ± 0.46 days. About complications, there were two (3.03%) unilateral recurrent laryngeal nerve lesions, one (1.52%) transient recurrent laryngeal nerve lesion and one (1.52%) permanent recurrent laryngeal nerve lesion. No cervical haematoma or wound infection was observed.No statistically significant difference was found in terms of the operative time, the use of intraoperative nerve monitoring, the use of drain, postoperative stay and complications between the two groups.p = 0.045).On the contrary, the use of the energy-based device was significantly greater in the HT Group than in the CT Group . However, other studies concluded that timing has no influence on morbidity ,37.The main aim of this study was to assess the complication rate in this category of patients, particularly in those undergoing a completion thyroidectomy after a hemithyroidectomy for a thyroid nodule with indeterminate cytology.Data for this analysis were collected from four high-volume thyroid surgery centres in Italy, with consequent high experience in this field of surgery.In order to eliminate the bias related to different surgical techniques, only patients undergoing a conventional open thyroidectomy were included, while patients submitted to a minimally invasive video-assisted thyroidectomy (MIVAT), transoral endoscopic thyroidectomy vestibular approach (TOETVA) and robot-assisted transaxillary thyroidectomy (RATT) were excluded. Moreover, in order to more accurately assess postsurgical hypoparathyroidism, patients simultaneously submitted to a parathyroidectomy were also not included.In our analysis, no statistically significant difference was found in terms of the occurrence of hypoparathyroidism, cervical haematoma, recurrent laryngeal nerve injury and wound infection comparing both the TT Group with the HT + CT Group  and the HT Group with the CT Group .Moreover, with regard to the secondary endpoint, which was the comparison of the HT Group with the CT Group in terms of operative time, no statistically significant difference was found. In this regard, it is important to underline that operative times were comparable even though the use of the energy-based device, which was well described in the literature as leading to a reduction in operative times, was slightly greater, but with a statistically significant difference, in the HT Group ,39.Regarding our results on complications and operative times, we believe they are justified by the fact that, in these patients, a completion thyroidectomy is performed on a “virgin” surgical field, free of anatomical distortions resulting from a previous operation, which does not entail any particular technical difficulty for the surgeon. Actually, it is important to note that, in addition to the completion thyroidectomy, a central neck dissection was performed in 19.70% of the patients, making it necessary to return to the side of the first surgery. However, despite this, the complication rate in patients who underwent a completion thyroidectomy was comparable to that of patients submitted to primary thyroid surgery .Finally, about the timing of the completion thyroidectomy, the complication rate was comparable even though the completion surgery was, on average, performed 2.12 ± 1.20 months after the first operation, thus in the so-called “intermediate” postoperative period. For this reason, as claimed by other authors, we do not believe that the timing between the two surgical procedures has an influence on the development of complications .As regards the rate of the completion thyroidectomy among patients undergoing a hemithyroidectomy, it was only 26.94%. Moreover, the completion surgery was not necessary in all patients submitted to a hemithyroidectomy with a subsequent histological finding of malignancy, but it was in 56.90%. Thus, following the ATA guidelines, unnecessary overtreatment, represented by total thyroidectomy, was avoided in a high percentage of patients. In this regard, it is also important to underline that a total thyroidectomy is burdened by a higher overall risk of complications, with specific reference to hypoparathyroidism and bilateral recurrent laryngeal nerve injury, which cannot occur in patients submitted to a hemithyroidectomy. Furthermore, as is well known and described by our data . Furthermore, also the result on operative times, obtained from the comparison between unilateral procedures (a hemithyroidectomy and a completion thyroidectomy), suggests that, in these patients, completion surgery does not entail any particular technical difficulty for the surgeon.Based on our findings, we believe that, when possible, in accordance with the American Thyroid Association guidelines, in patients with thyroid nodular disease with an indeterminate cytology, a hemithyroidectomy should always be performed as the initial surgery. This surgical strategy allows to avoid unnecessary overtreatment and the complications and overall costs associated with a total thyroidectomy, without an increased risk of complications in the case of a need for a completion thyroidectomy.Given the main limitation of our study, further prospective studies are needed to better investigate this topic.