Although around 80% of endometrial cancers are diagnosed at early stages and present with a 5-year survival rate exceeding 95%, patients with advanced and recurrent disease show a poor prognosis and low response rates to standard chemotherapy. In the era of targeted therapy, the great advances in the understanding of programmed death-ligand 1 (PD-L1) upregulation in cancer cells, which is responsible for tumor immune escape, have contributed to the increasing interest in immune checkpoint inhibitors as a promising strategy for the treatment of several refractory solid malignancies, including endometrial cancer. Several clinical trials have investigated the efficacy and safety of immune checkpoint inhibitors in endometrial cancer, which already led to the approval of the anti-programmed cell death protein 1 (anti-PD-1) antibody pembrolizumab as a satisfactory alternative for selected patients with unresectable or metastatic disease. As the future of cancer treatment will probably rely on combination therapy strategies, currently, innovative ongoing trials are exploring the potential role of immune checkpoint inhibitors associated with chemotherapy, radiotherapy, and other targeted therapies. Moreover, further research is warranted to discover new specific biomarkers that can accurately predict the response to immunotherapy.  Endometrial cancer (EC) is the most common gynecological cancer in developed countries, with a lifetime risk of 2.8% . The AmeThe human immune system consists of two main complementary subsystems: (1) the innate immune system, which gathers dendritic cells, natural killer (NK) cells, macrophages, neutrophils, eosinophils, basophils, and mast cells and does not depend on a prior stimulation by antigens , and (2)Historically, EC has been classified according to Bokhman’s model into two subtypes, based on clinicopathological and molecular features. The type I EC is the most common (60–70% of cases) and includes grades 1–2 endometrioid histology. It is associated with a high expression of estrogen receptors (ERs) and progesterone receptors (PgRs) and a good prognosis . On the other hand, the type II represents about 30–40% of all EC, typically embracing serous, clear cell or grade 3 endometrioid histology, and is estrogen-independent . It is tIn 2013, the Cancer Genome Atlas Research Network, given the new advances on the understanding of EC genomic landscape, introduced a new molecular classification that could change adjuvant treatment for women with an aggressive disease, based on four categories: polymerase ε (POLE)-ultramutated, microsatellite instability-high (MSI-H), copy-number low, and copy-number high . Each ofSeveral clinical trials have been published regarding the role of immune checkpoint inhibitors in endometrial cancer .In 2015, Le et al. first evaluated the clinical activity of pembrolizumab, an anti-PD-1 immune checkpoint inhibitor, in 41 patients presenting with progressive metastatic colorectal cancer (CRC) with or without MMR deficiency or other MMRd cancers. Pembrolizumab was administered intravenously (IV) at a dose of 10 mg/kg every 14 days in three different cohorts of patients, which included, respectively, MMRd CRC, MMRp CRC, and MMRd noncolorectal cancers, among which there were also 2 cases of EC. Co-primary endpoints were the immune-related objective response rate (ORR) and the 20-week immune-related progression-free survival (PFS). In the MMRd CRC group, the estimated ORR and the 20-week PFS were 40% and 78%, respectively, versus 0% and 11% reported for MMRp CRC patients. In the MMRd cohort including two EC patients, the ORR and PFS were 71% and 67%, respectively .In 2016, Mehnert et al. published the case of a single patient with a rapid and persistent (more than 14 months) clinical response to anti-PD-1 pembrolizumab treatment. The molecular analysis of a pretreatment biopsy revealed a POLE-ultramutated EC, thus suggesting that the identification of a POLE mutation might have a predictive value of treatment response to pembrolizumab. However, these data need to be further investigated to be confirmed . In 2017, Ott et al. published the results of the phase Ib KEYNOTE-028 trial, which enrolled 24 advanced EC patients treated with pembrolizumab 10 mg/kg IV administered every 2 weeks for a maximum of 24 months or until confirmed disease progression, intolerable toxicity, or death. The ORR reported was 13%. Three women showed a partial response, and among them, one had POLE mutations, whereas three patients achieved a stable disease. Six-month PFS and OS were 19.0% and 68.8%, respectively. Thirteen patients (54.2%) experienced treatment-related adverse events (TRAEs), such as fatigue (20.8%), pruritus (16.7%), pyrexia (12.5%), and decreased appetite (12.5%). Grade 3 adverse events (AEs) were reported in 4 patients, including asthenia, back pain, anemia, hyperglycemia, hyponatremia, pyrexia, and diarrhea. No patient experienced grade 4 AEs or any grade immune-mediated AEs, discontinued treatment because of an AE, or experienced treatment-related death. .In a recent phase II trial, Makker et al. tested the anti-PD-1 pembrolizumab at a dosage of 200 mg IV every 3 weeks in combination with lenvatinib, a multikinase-inhibitor of vascular endothelial growth factor (VEGFR), at a dosage of 20 mg daily orally in 108 advanced recurrent EC patients. The primary endpoint was the ORR at 24 weeks, according to the response evaluation criteria in solid tumors (RECIST) criteria 1.1. Forty-two (38.0%) patients had an objective response to lenvatinib plus pembrolizumab. The median PFS and overall survival (OS) were 7.4 months and 16.7 months, respectively. For patients with MSS/MMRp tumors, the ORR was 37.2%, whereas for patients with MSI-H/MMRd tumors, the ORR was 63.6%. The median duration of response (DOR) was not estimable (NE)  for patients with MSS/MMRp tumors and was 21.2 months  for patients with MSI-H/MMRd tumors. The most frequently reported any-grade AEs were hypertension (66 (61%)), fatigue (56 (51.9%)), diarrhea (57 (52.8%)), and hypothyroidism (48 (44.4%)). Grade 3–4 AEs occurred in 75 (69.4%) patients .Results from the Keynote-158 study demonstrated the clinical benefit of pembrolizumab for previously treated unresectable or metastatic MSI-H/MMRd noncolorectal cancer patients. Among 233 patients enrolled, 47 were EC patients. Eligible participants received pembrolizumab 200 mg IV every 21 days for 35 cycles or until documented disease progression, unacceptable toxicity, or patient/investigator decision. The primary endpoint was ORR, defined as the proportion of patients with confirmed complete/partial response per RECIST 1.1. An ORR of 57.1% was reported for EC patients, with a median PFS of 25.7 months. One hundred and fifty-one of 233 patients (64.8%) experienced TRAEs, and among them, 34 (14.6%) had grade 3–5 AEs. The most common toxicities were fatigue (14.6%), pruritus (12.9%), diarrhea (12.0%), and asthenia . Most frequent grade 3 AEs were increased gamma-glutamyltransferase (1.7%) and pneumonitis (1.3%). Three patients had grade 4 AEs: one patient had Guillain–Barre syndrome (a patient with gastric cancer), one had increased alanine aminotransferase (ALT), and another one had decreased neutrophil count and enterocolitis .In 2016, Santin et al. reported two cases of recurrent/metastatic EC, respectively a mixed clear cell and endometrioid (CC/EAC) POLE-ultramutated EC and a serous MSH6-mutated EC, not responding to surgery, chemotherapy, and radiotherapy and treated with nivolumab, a novel anti-PD-1 antibody, administered IV at a dosage of 3 mg/kg once biweekly. Both tumor samples evaluated in the pretreatment setting showed moderate peri- and intratumoral lymphocytic infiltrate  and moderate PD-1 and PD-L1 expression. Patients showed a persistent clinical response to nivolumab without severe toxicities, as confirmed by a computed tomography (CT) scan performed, respectively, at 7 and 9 months from the beginning of treatment .Preliminary data from the GARNET study were presented at the 2019 Society for Gynecologic Oncology (SGO) annual meeting on women’s cancer. One hundred and ten EC patients received dostarlimab 500 mg every 3 weeks. The ORR was 27.7% (50% in MSI-H and 19.1% in MSS). Sixty-eight EC patients (68%) experimented at least one TRAE. Grade ≥ 3 AEs were reported in 13 patients (11.8%), and the most common was increased serum levels of aspartate aminotransferase .Fleming et al. presented the results of a phase Ia clinical trial in which atezolizumab, an anti-PD-L1 antibody, was tested in 15 women with advanced and recurrent EC with microsatellite stability (MSS), MSI-H, or microsatellite unknown status. Patients received atezolizumab 1200 mg or 15 mg/mq IV in monotherapy every 21 days. Two women showed a partial response, while another two achieved a stable disease with an ORR of 13% and a disease control rate (DCR) of 27%. Both responders had a PD-L1 expression above 5% and presented, respectively, MSS and MSI-H status. It was reported a median PFS of 1.7 months and a median OS of 9.6 months. Only two patients experienced severe drug-related AEs (colitis and rash), whereas no G4–5 adverse events were recorded .Konstantinopoulos et al. published the results of a phase II trial, in which avelumab was tested in patients with MMRd/MMRp-recurrent or -persistent endometrial cancer. Thirty-three patients were enrolled. Avelumab was administered at 10 mg/kg IV every 2 weeks until progression or unacceptable toxicity. No patient with POLE-ultramutated tumor was enrolled in MMRd and MMRp cohorts. Co-primary endpoints were PFS at 6 months (PFS6) and ORR according to RECIST criteria 1.1. The MMRp cohort was closed at the first stage because only one out of 16 patients exhibited both ORR and PFS6 responses. The MMRd cohort achieved the primary endpoint of four objective responses after the accrual of only 17 patients. Of 15 patients who received avelumab, four exhibited an objective response  and 6  PFS6 responses . Responses were observed in the absence of PD-L1 expression. Twenty-two patients experienced TRAEs. Particularly, 6 patients reported grade 3 toxicity, including anemia, sinus bradycardia, hypothyroidism, diarrhea, myositis, and rash acneiform .Data from the PHAEDRA study was presented at the American Society of Clinical Oncology (ASCO) 2019 and showed the activity of durvalumab as a single agent in MMRd and MMRp cohorts. Seventy-one patients with advanced EC were enrolled: 35 patients with MMRp progressing after 1–3 lines of chemotherapy and 36 patients with MMRd progressing after 0–3 lines of chemotherapy. All patients received durvalumab at dosage of 1500 mg IV every 4 weeks. Among MMRd patients, the ORR was 40% , whereas within the MMRp group, the ORR was 3% . Adverse events occurred in 14 patients, including hyperthyroidism, hypothyroidism, pneumonitis, and hepatitis .Immune checkpoint inhibitors are under evaluation in several phase I, II, and III clinical trials, alone or in combination with other drugs . NCT0357Moreover, pembrolizumab is being investigated in phase I, II, and III clinical trials. NCT02549209 is a single-arm, open-label, multi-center phase II study in patients with newly diagnosed stage III/IV or recurrent EC that aims to assess the association of pembrolizumab with carboplatin/paclitaxel therapy. Patients with no prior therapy will receive pembrolizumab 200 mg plus carboplatin AUC 6 and paclitaxel 175 mg/mq every 21 days, whereas those with prior external beam radiation therapy (EBRT) and/or platinum-based chemotherapy must initiate paclitaxel and carboplatin at a reduced dose (paclitaxel 135 mg/mq plus carboplatin AUC 5). The primary endpoint is the ORR .The TOPIC study (NCT03276013) is non-randomized, single-arm, multi-center, phase II study of pembrolizumab in combination with doxorubicin in patients with recurrent/metastatic EC. Patients will receive doxorubicin 60 mg/kg IV every 3 weeks for up to 9 cycles in combination with pembrolizumab (MK-3475) 200 mg IV every 3 weeks. The primary endpoint is the PFS6 . NCT0351Two phase III clinical trials are evaluating the efficacy of pembrolizumab combined with other drugs for advanced or recurrent EC patients. In the randomized, placebo-controlled NCT03914612 study, patients will receive pembrolizumab every 21 days in association with carboplatin and paclitaxel for up to 6 cycles and then pembrolizumab every 3 weeks for up to 29 cycles as maintenance in the absence of disease progression or unacceptable toxicity . The ranMITO END-3 (NCT03503786) is a randomized, phase II clinical trial which compares carboplatin/paclitaxel to carboplatin/paclitaxel plus avelumab in advanced or recurrent EC. Patients in the experimental arm will receive carboplatin AUC 5, paclitaxel 175 mg/mq, and avelumab 10 mg/kg every 21 days for 6–8 cycles plus avelumab 10 mg/kg as maintenance every 14 days until disease progression or unacceptable toxicity. The primary endpoint is PFS .Furthermore, durvalumab is currently under evaluation in several clinical trials. NCT04269200 is a randomized, multicenter, double-blind, placebo-controlled, phase III study which aims to assess the efficacy and safety of durvalumab in combination with platinum-based chemotherapy (paclitaxel plus carboplatin) followed by maintenance durvalumab with or without olaparib for patients with newly diagnosed advanced or recurrent EC. In cohort A, patients will receive carboplatin/paclitaxel plus durvalumab placebo followed by maintenance durvalumab placebo and olaparib placebo; in cohort B, patients will undergo platinum-based chemotherapy and durvalumab followed by maintenance durvalumab and olaparib placebo; and in cohort C, they will receive platinum-based chemotherapy and durvalumab followed by maintenance durvalumab and olaparib. PFS is the primary endpoint .Finally, the EndoBARR trial (NCT03694262) is an open label, non-randomized multicenter, phase II trial evaluating the efficacy and safety of combined rucaparib, atezolizumab, and bevacizumab in recurrent and progressive EC. Atezolizumab will be administered at dosage of 1200 mg IV (every 3 weeks) together with bevacizumab 15 mg/mq (every 3 weeks) and rucaparib 600 mg twice daily. The primary endpoint is ORR .Understanding the spectrum of the unique toxicities associated with immune checkpoint inhibition is crucial, as they may be acute and life-threatening. The precise pathophysiology underlying immune-related AEs is unknown, although it appears related to the role that immune checkpoints play in maintaining immunologic homeostasis. As CTLA-4 and PD-1 agents act differently, their use causes different immune-related AEs. Generally, the PD-1 blockade seems to be better tolerated and has a lower AE frequency than the CTLA-4 blockade. Moreover, CTLA-4-related AEs increase with increasing dose whereas PD-1-related AEs seem not to be dose-related . OverallOver the last years, immunotherapy has meaningfully changed the paradigm of care of several solid tumors and, currently, it represents an active area of research aiming to identify which types of cancer could benefit most from it. Recently, the great advances in the understanding of cancer genome have contributed to the increasing interest in immune checkpoint inhibitors as a promising alternative strategy for refractory cancers ,64. Base


TP63, SOX2, ECT2, and PIK3CA. Furthermore, novel targetable oncogenic kinases within this amplicon include PRKCI, PAK2, MAP3K13, and TNIK. TCGA analysis of these genes identified amplification in more than 20% of clinical squamous cell carcinoma samples, correlating with a significant decrease in overall patient survival. Alteration of these genes frequently co-occurs and is dependent on 3q-chromosome amplification. The dependency of cancer cells on these amplified kinases provides a route toward personalized medicine in squamous cell carcinoma patients through development of small-molecules targeting these kinases.Squamous cell carcinomas of the lung, head and neck, esophagus, and cervix account for more than two million cases of cancer per year worldwide with very few targetable therapies available and minimal clinical improvement in the past three decades. Although these carcinomas are differentiated anatomically, their genetic landscape shares numerous common genetic alterations. Amplification of the third chromosome’s distal portion (3q) is a distinguishing genetic alteration in most of these carcinomas and leads to copy-number gain and amplification of numerous oncogenic proteins. This area of the chromosome harbors known oncogenes involved in squamous cell fate decisions and differentiation, including  Squamous cell carcinomas (SCCs) constitute a large proportion of all human cancers with little to no targetable therapies available . OutsideCDKN2A (p16), is a prominent genetic alteration in HPV- SCCs. The E6 and E7 HPV viral proteins inhibit the p53 and p105-RB proteins (binding partners of p16), respectively, in HPV+ tumors, thereby making genetic mutations of TP53 and CDKN2A uncommon in these carcinomas [The diagnosis and treatment of SCCs typically follows the anatomical tissue distribution of the cancer; for example, LSCC would be treated differently than HNSCC. However, SCCs are closely linked genetically, with mutations in cell-cycle proteins and increased expression of oncogenic molecules involved in squamous cell fate and/or differentiation pathways . FurtherCDKN2A p1, is a prrcinomas .TP63, SOX2, ECT2, and PIK3CA [Within both HPV+/− SCC tumors, a unifying genetic alteration in >75% of clinical samples is copy-number gain (CNG) (GISTIC score of +1) or amplification (GISTIC score of +2) of chromosome 3q 26-29 (3q amplicon), harboring key oncogenic drivers involved in SCC cell fate determination, including d PIK3CA . Althougd PIK3CA .PIK3CA and PRKCI, while providing evidence for novel druggable kinases including MAP3K13, TNIK, and PAK2. This review is intended to provide a better understanding of SCCs and highlight personalized treatment options targeting druggable kinases in SCCs.In this review, we will summarize the discovery of new druggable proteins, focusing on kinases within the 3q amplicon, and their application in the development of novel SCC treatments. We will highlight the current approaches taken toward treating these SCCs along with the experimental clinical trials on kinases being conducted. We review resident genes of the 3q amplicon including the oncogenic kinases LSCC is one of the most predominant forms of SCC, accounting for ~500,000 new cases per year . AlthougThere have been some minor steps toward the development of targeted therapies for LSCC . The firHNSCC is also a predominant form of SCC with >600,000 new cases each year ,13. MuchESCC is a highly fatal malignancy, accounting for ~500,000 cases each year with a five-year survival rate ranging from 15 to 20% ,15. ESCCCvSCC accounts for ~500,000 cases each year, predominantly in underdeveloped and developing countries (85% of deaths) . The devThe search for novel, antineoplastic therapeutic targets has been heavily aided by next-generation sequencing (NGS) advances. Projects such as The Cancer Genome Atlas (TCGA) have provided a wealth of genetic data for multiple types of cancers. Gene mutations that result in chronic proliferation, known as Mut-driver genes, have been identified in only 125 of the 20,000 protein-coding genes . All theThe EGFR (HER1) inhibitors, cetuximab, afatinib, and necitumumab, remain the only FDA-approved, first-line, targeted therapeutic for SCC to date. EGFR belongs to the ErbB family of receptor tyrosine kinases which comprises EGFR, HER2, HER3, and HER4. Many solid tumors have an upregulation of EGFR, including most LSCCs (>80%)  and HNSC2) producing phosphatidylinositol 3,4,5-trisphosphate . Class 1 consists of four different catalytic isoforms, p110α, β, γ, and δ, encoded by PIK3CA, PIK3CB, PIK3CG, and PIK3CD genes, respectively. PtdIns 3,4,5-P3 is a second-messenger lipid generated by PI3K, which activates several downstream signaling cascades. Effector proteins of PtdIns 3,4,5-P3 all contain a pleckstrin homology (PH) domain capable of binding PtdIns 3,4,5-P3. These effector proteins include, but are not limited to, AKT, phosphoinositide-dependent-kinase-1 (PDK1), Bruton’s tyrosine kinase, and guanine nucleotide-exchange factors  comprises the protein subunits rapamycin-insensitive companion of mTOR (RICTOR), mammalian stress-activated protein kinase interaction protein 1 (mSIN1), mammalian lethal with sec-13 protein 8 (mLST8), and protein observed with RICTOR 1 and 2 (Protor1/2) as well as phosphorylates AKT at S473 leading to the complete activation of AKT and results in an increase in cellular proliferation and survival. The tumor suppressor protein, PTEN, dephosphorylates PtdIns 3,4,5-P3 at the 3′ position yielding PtdIns-4,5-P2, thereby terminating the activation of downstream AKT signaling.Class 1 PI3Ks are the main class associated with cancer responsible for the phosphorylation of phosphatidylinositol 4,5-bisphosphate  is a nonreceptor tyrosine kinase that interacts with type I and type II cytokine receptors to activate the JAK-STAT pathway involved in cell proliferation and death . There aAtaxia telangiectasia and Rad3-related protein (ATR) is a serine/threonine protein kinase belonging to the phosphatidylinositol 3-kinase-related kinase (PIKK) family which includes five other members: ataxia telangiectasia mutated (ATM), DNA-dependent protein kinases (DNA-PKcs), mTOR, transformation/transcription domain-associated protein (TRRAP), and SMG1. ATR, along with ATM, are large sensory proteins that respond to DNA damage and regulate multiple essential cellular functions, including cell-cycle checkpoint activation and DNA damage repair. ATM is activated upon the detection of double-stranded DNA breaks, and loss of ATM protein expression is a common occurrence in cancer cells . ATR recLoss of ATR activity allows cells to avoid activation of cell-cycle checkpoints and continue with proliferation in the presence of DNA damage generated by chemotherapeutics. ATR inhibitors prevent the ATR-dependent DNA repair mechanisms induced by cisplatin chemotherapy, for example, and this potentiates the effect of the chemotherapy and results in mitotic catastrophe and cellular death . CurrentVascular endothelial growth factor receptor (VEGFR) is a well-studied receptor tyrosine kinase (RTK) with a biological function to regulate vasculogenesis and angiogenesis. There are three members of the VEGFR family: kinase insert domain-containing receptor (KDR/VEGFR-2), Fms-like tyrosine kinase (FLT1/VEGFR-1), and FLT4 (VEGFR-3). VEGFR-2 is the primary kinase receptor of VEGF-A for activation of angiogenesis, cellular proliferation, and survival. VEGFR-2 expression is greatly increased within the majority of solid tumors and serves as a vital signal transducer for cellular growth and survival, making it an intriguing pharmacological target. VEGF-A binding to VEGFR-2 activates a downstream proangiogenic signaling cascade, primarily through a PLCγ-PKC-MAPK axis . NumerouFibroblast growth factor receptors (FGFRs) comprise a family of four highly conserved receptor tyrosine kinases (FGFR1–4) and an additional receptor, FGFR5, which lacks an intracellular kinase domain. Receptor activation through the binding of fibroblast growth factor (FGF) activates a downstream signaling cascade mediated primarily through FGFR substrate 2 (FRS2) and PLCγ, resulting in cellular survival and proliferation through MAPK and PI3K/AKT signaling . Other pGains or amplifications of the distal region of the 3q chromosome, particularly 3q26-29 (3q amplicon), is a common genetic phenotype present in more than 75% of patients with LSCC, HNSCC, ESCC, and CvSCC, while also being prevalent in ovarian and uterine carcinomas ,9,10,41.PIK3CA, in addition to PRKCI, MAP3K13, TNIK, and PAK2. There is preferential amplification of these genes with at least one gene being amplified in more than 40% of LSCC, 35% of ESCC, 19% of CvSCC, and 15% of HNSCC compared to other cancers  is a member of the atypical protein kinase C isozymes coded by the ylserine ,46,47. Rylserine . PKCι exylserine ,50,51,52ylserine ,54,55. Fylserine . In addiylserine ,58.p-value < 0.001, LOG2 > 3)  . Small-mThe leucine zipper-bearing kinase (LZK) encoded by the MAP3K13 gene is an understudied protein kinase belonging to the mixed lineage kinase (MLK) family. LZK was first identified as a regulator of the c-Jun N-terminal kinase (JNK) pathway through phosphorylation of MAP2Ks (MKK4 and MKK7) ,61. LZK Our group recently demonstrated that LZK is essential for the survival of HNSCC cell lines harboring the 3q amplicon. LZK mediates its oncogenic function independent of JNK and NF-κB signaling and utilizes a novel mechanism to stabilize mutant gain of function (GOF) p53, thereby regulating AMP-activated protein kinase (AMPK) and p21 expression.  AnalysisTraf2-and NCK-interacting kinase (TNIK) was first identified as cytoskeletal regulating protein that activates the JNK pathway . It was TCGA analysis shows TNIK has an important function in SCCs and correlates with a significant decrease in patient survival when there are amplifications of the gene . Amplifip21-activated kinase 2 (PAK2) was initially identified as an RHO GTPase, CDC42, and an RAC1 binding partner . PAK2 isAnalysis of PAK2 utilizing cBioPortal within LSCC, HNSCC, ESCC, and CvSCC shows amplification of PAK2 in 21% of the clinical samples . AmplifiSCCs of the lung, head and neck, esophagus, and cervix continue to be global health concerns with little development in targeted therapies over the last three decades. Current steps toward developing therapies for SCCs largely involve either immunotherapy  or kinase inhibitors. Promising clinical trials are being conducted on kinase inhibitors that target the squamous cell growth and differentiation pathways, including upstream receptor tyrosine kinases  and PI3K . HoweverTP63, SOX2, ECT2, and PIK3CA. Numerous research groups have identified other potential oncogenes in this region, including FXR1, CLAPM1, EIF4G, SEC62, and SKIL [A unifying genetic alteration in all SCCs is the distal amplification of the 3q chromosome resulting in amplifications of key genes involved in squamous cell fate decisions and differentiation, including and SKIL ,80,81,82and SKIL . FurtherPIK3CA, PRKCI, MAP3K13, TNIK, and PAK2 as exciting, plausible therapeutic targets for SCCs. Numerous studies have demonstrated that multiple different cancers both within and outside of SCCs depend on PKCι . Furtheiewed in , and ouriewed in ,70. Coll


The loss of skeletal muscle mass (muscle atrophy or wasting) caused by aging, diseases, and injury decreases quality of life, survival rates, and healthy life expectancy in humans. Although long non-coding RNAs (lncRNAs) have been implicated in skeletal muscle formation and differentiation, their precise roles in muscle atrophy remain unclear. In this study, we used RNA-sequencing (RNA-Seq) to examine changes in the expression of lncRNAs in four muscle atrophy conditions  in mice. We successfully identified 33 annotated lncRNAs and 18 novel lncRNAs with common expression changes in all four muscle atrophy conditions. Furthermore, an analysis of lncRNA–mRNA correlations revealed that several lncRNAs affected small molecule biosynthetic processes during muscle atrophy. These results provide novel insights into the lncRNA-mediated regulatory mechanism underlying muscle atrophy and may be useful for the identification of promising therapeutic targets. In the last decade, increasing evidence has indicated that long non-coding RNAs (lncRNAs), defined as having more than 200 bases in length, have important roles in cell proliferation and differentiation, development, and the maintenance of homeostasis via interactions with essential proteins . AccordiAtrolnc-1 in catabolic conditions promotes muscle atrophy by increasing NF-κB activity [Pvt1 is associated with muscle atrophy [Myoparr, which is expressed from the promoter region of the myogenin gene, promotes muscle atrophy after denervation treatment by activating myogenin expression and inhibiting BMP signaling. The inhibition of Myoparr attenuates the decrease in muscle mass caused by denervation [Skeletal muscle is a mechanically pivotal organ in humans; it is required for movement of body parts and the body as a whole. Skeletal muscle is also a highly plastic organ, and its mass decreases through aging, immobilization, malnutrition, and diseases, including cancer, cardiovascular diseases, and neurodegenerative diseases ,11,12,13activity . In a mo atrophy . We haveervation ,22. ThusDRR, DUM1, linc-MD1, linc-YY1, LncMyod, Neat1, Myoparr, Malat1, and SRA) and genomic imprinting-related lncRNAs  [We have recently shown that muscle atrophy conditions can be classified into two sub-groups (disuse-mediated atrophy and systemic wasting atrophy) based on the expression levels of skeletal muscle differentiation-related lncRNAs ( IG-DMR) . It is h IG-DMR) ,24. In tWe performed an RNA-Seq analysis to identify protein-coding mRNAs and lncRNAs whose expression was altered across multiple skeletal muscle atrophy conditions in mice . Based on the expression profiles of both mRNAs and lncRNAs revealed by RNA-Seq, muscle atrophy conditions could be classified into two sub-groups: disuse-mediated atrophy, including the denervation and casting treatments, and systemic wasting atrophy, including fasting and cancer cachexia treatments A. These To further characterize the identified mRNAs and lncRNAs, their structural features were examined. The chromosomal distribution of the identified mRNAs and lncRNAs did not differ significantly, except that only lncRNAs were distributed on chromosome Y and mitochondria A. The lnNext, to clarify the signaling pathways involved in each type of muscle atrophy, we examined the identified mRNAs using a functional enrichment analysis. The upregulated mRNAs, in response to denervation, were preferentially related to rRNA processing, whereas mRNAs that were down-regulated in denervated muscles were enriched in muscle system processes A,B. The Cdkn1a and Ctsl) [Abcc8, Csrp3, Dlg5, Gdf5, Gpnmb, Il4ra, and Runx1) [It is likely that mRNAs with altered expression across all four muscle atrophy conditions are involved in the development or progression of skeletal muscle atrophy. We identified 71 and 122 mRNAs whose expression levels were commonly increased and decreased in all muscle atrophy conditions, respectively . We evalnd Ctsl) ,26 and pd Runx1) ,32,33,34d Runx1) ,37,38,39We next extracted lncRNAs that were up- or down-regulated in all four atrophy conditions. Five and 28 annotated lncRNAs were up- and down-regulated in all muscle atrophy conditions, respectively . Seven ulinc-Myh (2310065F04Rik) and Gm49794) and down-regulated unannotated lncRNAs (G408 and G10427), were related to changes in mRNAs involved in small molecule biosynthetic processes  and 193 mRNAs (71 up-regulated and 122 down-regulated mRNAs) and following functional enrichment analysis of correlated mRNAs showed that the expression changes of four identified lncRNAs, including down-regulated annotated lncRNAs  were evaluated in each muscle atrophy condition. The quantification results obtained by qPCR were almost identical to the results of the RNA-Seq analysis except for the expression of 2310015D24Rik in the casting model . We performed a quantitative analysis of multiple identified lncRNAs, including the four lncRNAs extracted from the lncRNA–mRNA network analysis. It should be noted that ng model A–F. For nificant A–D. The nificant E–L. ThesRecent global transcriptome analyses have identified numerous lncRNAs involved in the regulation of skeletal muscle formation and differentiation, indicating the importance of lncRNAs in myogenesis . HoweverOip5os1 and linc-Myh were substantially decreased in all muscle atrophy conditions. Oip5os1, also known as OIP5-AS1/cyrano, was initially implicated in the development of the brain and eye by morpholino studies in zebrafish [Oip5os1 mutant mice and zebrafish exhibited normal development [Oip5os1 contributed to myogenesis [Oip5os1 expression increased the production of Mef2c protein, a myogenic transcription factor, by recruiting HuR to the 3′utr of Mef2c mRNA [Mef2c mRNA levels decreased significantly after fasting and cachexia  via the INO80 chromatin remodeling complex [linc-Myh-knockout mice. Further analyses will reveal the precise role of the decreased expression of linc-Myh in muscle atrophy. Intriguingly, in our co-expression network analysis of lncRNAs and mRNAs, we identified four lncRNAs, linc-Myh, Gm48065, Gm49794, and G10427, that were involved in small molecule biosynthetic processes. Although the exact role of this pathway in the regulation of skeletal muscle mass remains unclear, it is noteworthy that Slc39a14, Mt1, and Mt2 in this pathway are associated with muscle mass [Slc39a14 (Zip14) encodes a zinc transporter, and its expression in skeletal muscle tissues is highly activated in response to lipopolysaccharide-induced inflammation. In mice, Slc39a14 knockout leads to muscle atrophy and metabolic endotoxemia, indicating that this gene is required to maintain skeletal muscle mass [Mt1 and Mt2, which encode metallothioneins, show muscle hypertrophy with increased muscle strength via the activation of the IGF-1/Akt/mTOR pathway [Molecular functions for most of the lncRNAs identified in this study are still unknown. However, the expression levels of ebrafish . Howeverelopment . More reogenesis . After mf2c mRNA . We obsecachexia . Mef2 wa in mice . Howeverertrophy . Conside complex , an incrcle mass ,47. Slc3cle mass . HoweverMSTRG.42019 was associated with skeletal muscle fiber-type switching in pigs [LncMyoD, SAM, H19, and CTTN-IT1, affect the activity of satellite cells in mice, pigs, and sheep [Pvt1 results in increases in the size and number of mitochondria [Skeletal muscle atrophy is the main physiological change common to all four models used in this study. However, the four models also show differences in muscle fiber-type, stem cell activity, and mitochondrial function. For example, denervation and casting induce a slow-to-fast fiber-type shift, whereas cancer cachexia and fasting cause a fast-to-slow fiber-type shift . In addi in pigs . Severalnd sheep ,55,56,57Mice (C57BL/6J and CD2F1) were purchased from the Japan SLC and Charles River Laboratories Japan. Mice were housed in cages with a constant temperature (24 °C) and a 12 h light: 12 h dark cycle and were provided water and food ad libitum. During the fasting experiments, no food intake was allowed. C57BL/6J mice were used for denervation, atrophy by casting and fasting, and CD2F1 mice were used for cachexia treatment. The detailed protocols for each muscle atrophy experiment and the changes in the skeletal muscle weight were described previously .RNA-Seq libraries were prepared as described previously . In briehttps://www.bioinformatics.babraham.ac.uk/projects/fastqc/ 26 February 2021) with the command “-Q 33 -t 20 l 30.” The reads were aligned to the GENCODE GRCm38_p6_genome using Hisat2 v. 2.2.0 [p-value, padj) < 0.05, and log2 fold change of >1 or <−1).Raw sequence data were quality-trimmed using FastQC ver. 0.11.3  . In thisytoscape .Rpl26 expression). Values are shown as each control -injected control mice, or muscles of mice provided water and food ad libitum) is 1. The primers are listed in t-tests. A value of p < 0.05 was considered statistically significant.One microgram of total RNA was used to generate cDNAs by reverse transcription using Protoscript II Reverse Transcriptase  with random primers. Quantitative PCR was performed using SYBR Premix Ex Taq . Relative gene expression was determined using a ΔΔCt method and normalized to a reference gene (


Meanwhile, high levels of Sirt3 and SOD2 induced by MSCs reduced the expression levels of IL-1β, TNF-α, ICAM-1, and MMP9 by suppressing the NF-κB/HMGB1/NLRP3/caspase-1 signaling pathway, as well as regulating the expression levels of cleaved caspasese-3, Bax, and Bcl2 by upregulating the expression level of P-Akt, thereby inhibiting the apoptosis of the lung tissue. In addition, MSCs also regulated the expression levels of LC3, P62, BiP, Chop, and PERK, thereby enhancing autophagy and attenuating endoplasmic reticulum stress. Taken together, our results suggest that MSCs effectively attenuate diabetic lung fibrosis via adjusting Sirt3-mediated responses, including inflammation, oxidative stress, apoptosis, autophagy, and endoplasmic reticulum stress, providing a theoretical foundation for further exploration of MSC-based diabetic therapeutics.Diabetes affects a variety of organs such as the kidneys, eyes, and liver, and there is increasing evidence that the lung is also one of the target organs of diabetes and imbalance of Sirt3-mediated stress responses such as inflammation, oxidative stress, apoptosis, autophagy, and ER stress may contribute to diabetic lung fibrosis. Although previous studies have reported that mesenchymal stem cells (MSCs) have beneficial effects on various diabetic complications, the effect and mechanisms of MSCs on diabetes-induced lung injury are not clear. In this study, the STZ-induced diabetes model was constructed in rats, and the effect and potential mechanisms of bone marrow MSCs on diabetic lung fibrosis were investigated. The results revealed that fibrotic changes in the lung were successfully induced in the diabetic rats, while MSCs significantly inhibited or even reversed the changes. Specifically, MSCs upregulated the expression levels of Sirt3 and SOD2 and then activated the Nrf2/ARE signaling pathway, thereby controlling MDA, GSH content, and iNOS and NADPH oxidase subunit p22 Diabetes mellitus (DM) is a chronic metabolic disease characterized by persistent blood hyperglycemia; it causes profound long-term effects on a variety of organs, such as the kidneys, heart, brain, eyes, and liver –5. In reκB pathway, TGF-β pathway, autophagy, apoptosis, and endoplasmic reticulum (ER) stress [+-dependent deacetylase; it is a key regulator of the mitochondrial respiratory chain and plays an important role in the pathophysiology of various diseases, such as diabetes and metabolic syndrome, and aging [It has been recognized that diabetes can induce various pathological responses, one of which is the development of fibrosis . Tissue ) stress –22. Sirtnd aging . Existinnd aging –26.κB-dependent inflammatory pathway in the proteinuric kidney disease [β-cells from ER stress-induced apoptosis and dysfunction [In diabetes pathogenesis, Sirt3 plays a protective role and involves a variety of stress responses. For example, Sirt3 could ameliorate oxidative stress and mitochondrial dysfunction after intracerebral hemorrhage in diabetic rats , alter t disease , and pre disease . Recent  disease  and protfunction . Thus, SMSCs harbor differentiation potential, immunosuppressive properties, and anti-inflammatory effects and are considered an ideal candidate cell type for the treatment of DM . MSCs arMale Sprague-Dawley rats were purchased from Chengdu Dossy Experimental Animal Co. Ltd. . All experiments involving animal subjects were performed in accordance with guidelines approved by the Animal Care and Use Committee of West China Hospital, Sichuan University.2 in a humidified atmosphere at 37°C, identified as described previously [To generate bone marrow MSCs, bone marrow mononuclear cells were harvested by flushing the tibiae and femurs of 3-week-old male Sprague-Dawley rats (50–55 g body weight) with phosphate-buffered solution (PBS). Bone marrow MSCs were cultured in complete medium   and grown with 5% COeviously . Bone man = 6 in each group). For the rats in the DM+BMSC group, 5 × 106 MSCs were suspended in 1 mL PBS and injected via the tail vein 6 times at a one-week interval. The rats in the DM+PBS group were infused with 1 mL PBS. One week after the last treatment of MSCs, all rats were sacrificed by cervical decapitation, and blood and lung samples were collected for further assessment.Eight-week-old male Sprague-Dawley rats were used in this study. Before the experiments, the rats were housed for 1 week to adapt to the experimental animal facility with an ambient temperature of 22–25°C and were allowed access to water and food. A total of 18 rats, of which 12 were grouped for diabetes induction and 6 animals were grouped as controls, were used. The diabetic rat model was induced by a single intraperitoneal injection of STZ  dissolved in the sodium citrate buffer at 55 mg/kg body weight into rats after overnight fasting. Control rats received an equal volume of citric acid buffer. One week after the injection, rats with fasting blood glucose contents exceeding 16.7 mM were deemed diabetic. Six weeks after the induction, the diabetic rats were randomly divided into diabetes mellitus+phosphate-buffered saline (DM+PBS) and diabetes mellitus+bone marrow MSC (DM+BMSC) groups .The activities of malondialdehyde (MDA) and micro reduced glutathione (GSH) in lung tissue were determined using an MDA Detection Kit  and a Micro Reduced GSH Assay Kit  according to the manufacturer's protocols.μm. The sections were stained with hematoxylin and eosin (H&E), Masson, Periodic acid-Schiff (PAS), and Sirius Red.For histological examination, rat lung tissue was fixed in 10% neutral-buffered formalin for 48 h, paraffin-embedded, and sectioned at an average thickness of 5 2O2, and unspecific binding sites were blocked with normal goat serum (Solarbio). The sections were incubated with primary antibodies against α-SMA, HMGB1, MMP9, P65, NLRP3, GPX-1, GST, Nrf2, and fibronectin overnight at 4°C. Then, the sections were incubated with a biotin-labeled secondary antibody followed by incubation with HRP-labeled streptavidin. Thereafter, the signals were detected with 3,30-diaminobenzidine, counterstained with hematoxylin, and observed under an optical microscope .Paraffin sections were dewaxed in xylene and rehydrated with decreasing concentration of ethanol, followed by antigen retrieval in 0.01 mol/L citrate buffer. Endogenous peroxidase was quenched with 3% HApoptosis was assessed in paraffin-embedded lung sections with the use of a terminal deoxynucleotidyl transferase dUTP nick end labeling  in situ cell death detection kit according to the manufacturer's instructions.β-actin.The total protein samples from lung tissues were homogenized using RIPA lysis buffer containing protease inhibitors  and phosphatase inhibitors . The protein concentrations of the samples were determined using the BCA Protein Assay Kit (Beyotime). The protein samples were loaded onto the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel (10–12.5%), separated electrophoretically, and transferred onto polyvinylidene difluoride membranes . After blocking with 5% nonfat milk for 1 h, the membrane was individually incubated overnight at 4°C with the primary antibodies listed in post hoc analysis or Kruskal-Wallis H with Student-Newman-Keuls (SNK) post hoc analysis. Statistical significance was defined as P ≤ 0.05.All statistical analyses were performed using GraphPad Prism v8.0  and expressed as the mean ± standard deviation (SD). All data were assessed for normality of distribution using the Kolmogorov-Smirnov and Shapiro-Wilk tests before data analysis, and differences between groups were analyzed using one-way ANOVA with Tukey's Lung tissue collagen content was analyzed by Masson staining and Sirius Red staining . Collageα-SMA, collagen I, collagen III, fibronectin, and cleaved TGF-β1, with concomitant decreases in the levels of E-cadherin, compared to those in the control rat tissue; meanwhile, compared with the DM+PBS group, these changes were significantly inhibited or even reversed in the DM+BMSC group. In addition, the immunohistochemistry assay also showed that the expression levels of α-SMA and fibronectin were markedly decreased in the DM+BMSC group than in the DM+PBS group  and fibrosis-associated biomarkers in lung tissues. As shown in Figures BS group .It is well known that the sirtuin family is composed of seven members (Sirt1 to Sirt7), out of which Sirt1, Sirt2, Sirt3, and Sirt6 exert positive effects on tissue fibrosis, especially Sirt3 , 42. To Diabetes is a chronic metabolic disease characterized by hyperglycemia, which is always accompanied by elevated blood triglyceride and cholesterol levels. Microenvironment with high fat and high glucose usually causes oxidative stress. Oxidative stress is implicated as an important molecular mechanism underlying fibrosis in a variety of organs, including the lungs. To assess the effects of MSCs on diabetes, changes in blood glucose, triglycerides, and total cholesterol were measured. As shown in phox in the lung tissues reflect the degree of oxidative stress. Higher levels of MDA, iNOS, NADPH oxidase subunit p22phox and lower levels of GSH were found in the DM+PBS group as compared to the control group  is a basic region leucine-zipper transcription factor that binds to the antioxidant response element (ARE) and thereby regulates the expression of a large battery of genes involved in the cellular antioxidant and anti-inflammatory defense. Immunohistochemistry and western blot analyses showed that the protein levels of Nrf2, glutathione-S-transferase pi (GST Pi), and glutathione peroxidase 1(GPX-1) significantly increased in the DM+MSC group compared with the DM+PBS group Figures , suggestHE and PAS staining of lung tissues was performed to evaluate lung injury. Rats in the control and the DM+BMSC groups showed normal lung structures with thin alveolar walls and light PAS staining . In contα, ICAM-1, IL-1β, and MMP9 were also significantly lower in the DM+BMSC group than in the DM+PBS group. Furthermore, the cytoplasmic translocation of HMGB1, the nuclear translocation of NF-κB p65, and the NF-κB/HMGB1/RAGE/NLRP3/caspase-1 signaling pathway were significantly inhibited by MSCs compared with the DM+PBS group , TNF-α, IFN-γ, matrix metalloproteinases (MMPs) and transforming growth factor β (TGF-β), and transcription factors such as nuclear factor-κB (NF-κB) [κB activation [β and IL-18. Recent reports have shown that NLRP3 and ASC/caspase-1/IL-1β signaling are important for HMGB1 induction and release, although the exact mechanism is unclear [κB [κB, NLRP3, and caspase-1 and upregulation of the Sirt3 expression inhibited the activation of NF-κB, HMGB1, NLRP3, and caspase-1 [κB p65/NF-κB p65, HMGB1, RAGE, NLPRP3, and cleaved caspase-1 and then significantly inhibited the levels of IL-1β, TNF-α, ICAM-1, and MMP9. These data indicate that MSCs increase Sirt3 expression and then inhibit the activation of the NF-κB/HMGB1/NLRP3 pathway to improve inflammatory response to diabetic lung fibrosis.Lung fibrosis is characterized by inflammatory and fibroproliferative changes, including the release of inflammatory cytokines such as interleukins (IL-1 (NF-κB) . These i (NF-κB) . High-mo (NF-κB) . Extracetivation . The NOD unclear –68. Anotlear [κB . Recent aspase-1 , 70, 71.Apoptosis plays an important role in the pathophysiology of DM , 73 and Autophagy is a molecular mechanism that maintains cellular physiology and promotes survival. Defects in autophagy lead to the causes of many diseases, such as diabetes mellitus, cancer, neurodegeneration, and infection disease, and to aging . RecentlEndoplasmic reticulum (ER) stress is associated with the development and progression of fibrotic diseases, including lung fibrosis . The glu+ mesenchymal cells (endogenous MSCs) served as a reservoir for epithelial cell regeneration and reestablishment of the normal airway epithelium during mouse lung LPS and naphthalene injury repair [In addition, the role of endogenous lung mesenchymal stem cells (lung MSCs) in lung injury repair has received increasing attention in recent years . An intey repair . AlthougMoreover, there are also several limitations to this study. First, in the present study, we have observed significant reduction in protein expressions of Sirt1, Sirt3, and Sirt6 in the DM+PBS group, except Sirt2, compared with the control rats. This observation might support the hypothesis that Sirt1, Sirt3, and Sirt6 could play an important role in the development of tissue fibrosis induced by diabetes, which is consistent with previous research results , 41, 42 In conclusion, our study demonstrates that MSCs effectively ameliorate diabetic lung fibrosis via regulating Sirt3-mediated stress responses, including enhancement of autophagy and inhibition of oxidative stress, inflammation, apoptosis, and endoplasmic reticulum stress. These results not only shed new light on the pathogenesis of diabetic lung injury but also provide a theoretical foundation for further exploration of diabetic lung fibrosis therapeutics.


Class-3 semaphorins (SEMA3s), initially characterized as axon guidance cues, have been recognized as key regulators for immune responses, angiogenesis, tumorigenesis and drug responses. The functions of SEMA3s are attributed to the activation of downstream signaling cascades mainly mediated by cell surface receptors neuropilins (NRPs) and plexins (PLXNs), yet their roles in human cancers are not completely understood. Here, we provided a detailed pan-cancer analysis of NRPs and PLXNs in their expression, and association with key signal transducers, patient survival, tumor microenvironment (TME), and drug responses. The expression of NRPs and PLXNs were dysregulated in many cancer types, and the majority of them were further dysregulated in metastatic tumors, indicating a role in metastatic progression. Importantly, the expression of these genes was frequently associated with key transducers, patient survival, TME, and drug responses; however, the direction of the association varied for the particular gene queried and the specific cancer type/subtype tested. Specifically, NRP1, NRP2, PLXNA1, PLXNA3, PLXNB3, PLXNC1, and PLXND1 were primarily associated with aggressive phenotypes, whereas the rest were more associated with favorable prognosis. These data highlighted the need to study each as a separate entity in a cancer type- and subtype-dependent manner. Class-3 semaphorins (SEMA3s), including seven members (SEMA3A-SEMA3G), are the only family of secreted proteins among all the ~28 identified vertebrate semaphorins. Semaphorins are characterized by the presence of a ~500 amino acids long conservative N-terminal sema domain signature which is also present in semaphorin receptors of the plexin family . SimilarNRPs are multifunctional, non-tyrosine kinase cell surface glycoproteins expressed in all vertebrates and widely conserved across species . The twoFor PLXNs, which like NRPs are also type 1 transmembrane proteins, semaphorins are their primary ligands. PLXN family contains nine members that are subgrouped into four subfamilies based on their structural homology: four Type-A PLXNs (PLXA1-A4), three Type-B PLXNs (PLXNB1-B3), and single C (PLXNC1) and D (PLXND1) PLXNs . To dateIn this study, we used a similar systematic pan-cancer analyses method to our study of SEMA3s to elucidate the function of NRPs and PLXNs in 33 different cancer types . Our resp < 0.0001), PLXNA1, PLXNA3, and PLXNB3 showed a significant increase (p < 0.0001), while NRP2, PLXNB1, PLXNB2, and PLXNC1, SEMA3A and SEMA3F showed no overall significant difference in tumors compared to that of normal samples averaged across all 33 cancer types (p > 0.05) (p < 0.0001). Interestingly, even the genes that showed no differential expression in tumors compared to normal samples also showed significantly increased (NRP2 and PLXNC1) or decreased (PLXNB1 and SEMA3F) expression in metastatic tumors (p < 0.0001). These results suggest that these genes play crucial roles not only during tumorigenesis but also during tumor metastasis  A. Notabltastasis A.p > 0.05) and they were not clustered together  , ovarianPLXNs need to activate PLXN-associated tyrosine kinases (TKs) or interact with intracellular signal transducers R-Ras or Rho family GTPases to transduce semaphorin-dependent signaling pathways ,35. Therp-value < 0.05 without adjustment for multiple comparisons to be consistent with the display of the results with forest plots  pan-cancer data, six types of immune infiltrates were identified across all cancer tumors that correspond from tumor promoting to tumor suppressive . They arr = 0.59), followed by PLXND1 (r = 0.54), NRP2 (r = 0.50), PLXNC1 (r = 0.48) and PLXNA4 (r = 0.28) (p < 0.0001). For immune score, PLXND1 showed the highest correlation (r = 0.46) across all cancer types, followed by PLXNC1 (r = 0.39), NRP1 (r = 0.23) and NRP2 (r = 0.15) (p < 0.0001). The order of the correlation between the expressions of NRPs and PLXNs and estimate score is the same as that for stromal score, but with different correlation coefficients   . Therefor: −0.65 to −0.13) (p < 0.0001), where NRPs showed the strongest association  across cancer types. For the correlation with DNAss score, PLXNA1 and A2 showed positive correlation , while all other members showed negligible (|r| < 0.17) or insignificant correlation (PLXNA3 and NRP2) across all cancer types. At the individual cancer type level, we found that NRP2 and PLXNA2 were positively, while PLXNB1 and C1 were negatively correlated with ovarian cancer stemness score measured with DNAss, but not with RNAss. In pheochromocytoma & paraganglioma (PCPG), PLXND1 was negatively, while PLXNB1 and B3 were positively correlated with both RNAss and DNAss. In addition, similar to what we observed for SEMA3s [Cancer stem cells (CSCs) have been considered responsible for tumor recurrence, metastasis, and chemotherapy resistance . A couplr SEMA3s , all genr SEMA3s .Z-scores, and the higher the score, the more sensitive the cells are to the drug treatment [Z-scores, we found that members of NRPs and PLXNs showed strong correlation with drug responses (|r| ≥0.4 and p < 0.0001), especially NRP1, NRP2, PLXNA1, PLXNA2, PLXNB1, PLXNB2, PLXNB3, and PLXNC1, although the direction of the correlation varies for different genes with different drugs   A. We furn = 7) B. In conn = 7) A,B. For n = 7) . In summp < 0.0001). Except PLXNA3, B1, B2, and B3 which was either not associated or negligibly associated with the three scores in TME, i.e., immune score, stromal score, and tumor purity score, other receptors were all positively correlated with the three scores (p < 0.0001) in breast cancer. NRP1, NRP2, PLXNC1 and D1 showed the strongest correlation (r > 0.35). These results were also similar to what was observed by using all the TCGA pan-cancer tumor data  (r < 0.12)  was associated with worse prognosis for PFI, which could be due to their higher expression either in aggressive molecular subtypes (PLXNA1 and PLXNA2) or immune infiltrate subtypes  or high association with tumor infiltrates  A–C. Incr 0.0001) . In cont < 0.12) B. These The semaphorin/neuropilin/plexin complexes control a wide range of biological processes and deregulation of these complexes is associated with multiple pathological statuses. In recent years, their roles in tumor growth, metastatic spread, and drug resistance are increasingly recognized. They have been found to be involved in controlling tumor cell viability, apoptosis, proliferation, adhesion, migration, and invasion by influencing both the tumor compartment and their microenvironment. Our previous study on the comprehensive characterization of SEMA3s showed that SEMA3A, SEMA3C, SEMA3E, and SEMA3F are more likely to promote tumorigenesis and associate with poor prognosis, while the other SEMA3s are more likely to play a tumor suppressor role and generally associate with better prognosis . Due to By including the gene expression analysis of SEMA3s with their receptors in this study, we noticed that the expression of the ligand is not necessarily positively correlated with that of their receptors. For example, PLXND1 is the receptor for SEMA3C, 3D, and 3E ,29,30,31Similar to SEMA3s, the expression of the receptors not only showed great heterogeneity among different tumor types, but also showed high heterogeneity within each histological subtype/tissue of origin, as well as in different immune subtypes. Our results demonstrated that the majority of the receptors showed great expression variation in different molecular subtypes of BRCA, HNSC, BLCA, OV and KIRC. At the individual gene and cancer type level, PLXNA1 and PLXNA3 were found to be predominantly up-regulated, PLXNA4 was predominantly down-regulated, while NRPs and the rest of the PLXNs showed mixed up- and down-regulation in the 17 tested cancer types, although overall NRP1 expression was downregulated, and PLXNB3 was upregulated averaged across all the 33 cancer types. Strikingly, almost all the NRP and PLXN members had more dramatic expression in metastatic tumors than in primary tumors in the pan-cancer data, indicating they play important roles not only during tumorigenesis but also during tumor metastatic progression. In fact, SEMA3E and its receptor PLXND1 was reported to promote tumor invasiveness and metastatic spreading of melanoma in mice, and SEMA3A was found to suppress tumor growth and metastasis in melanoma ,76. SEMAThe dysregulated expression of NRPs and PLXNs in cancer tumors was generally associated with patient overall survival and progression free interval in 33 cancer types, while the direction of association is dependent on the cancer type tested and the genes queried. In general, the upregulated gene expression was associated with worse survival, while the downregulated expression was associated with better prognosis. For example, PLXNA1 and PLXNA3, which were mainly upregulated in tumors, were mainly associated with poor prognosis, while NRPs, PLXNB3, PLXNC1 and PLXND1 had mixed association with survival (both advantage and disadvantage) that is cancer type specific, however, majority of the significant survival association were toward poor prognosis. The rest of the PLXNs, i.e., PLXNA2, A4, B1 and B2 were more associated with better survival. However, in some circumstances, we observed that decreased gene expression in cancer tumors associated with increased survival risk and vice versa which could be explained by the fact that the expression of NRPs and PLXNs were also molecular subtype- and immune infiltrate subtype-dependent within the same tumor origin. Regardless of the direction of the association, notably, almost all the members of NRPs and PLXNs were associated with both OS and PFI in UVM, which is worth further investigation.SEMA3s and their receptors NRPs and PLXNs are a new class of immunoregulatory molecules with distinct functions in various phases of the immune responses, which have been reported to play important roles in cancer-associated immune responses ,43. By sCancer stem-like-cells (CSC) promote cancer progression due to the capacity for self-renewal and invasion, and it is the main cause of treatment-induced drug resistance ,80,81. HBy studying NRPs and PLXNs in breast cancer in detail, we observed that expressions of these genes vary in different molecular subtypes and immune subtypes within the same tumor origin and confirmed that the tumor suppressor or promoter role could be subtype specific. For instance, even though genes PLXNB1, PLXNB3, and PLXND1 showed no significant differential expression in breast cancer tumors compared to normal, their expression was associated either with less aggressive breast cancer subtypes (PLXNB1) or with more aggressive subtypes or had further deregulated expression in metastatic tumors (PLXNB3 and PLXND1). This is corresponding to their prediction of patient survival outcomes with PLXNB1 showing favorable prognosis, while PLXNB3 and PLXND1 show poor prognosis for both OS and PFI in breast cancer tumors.In summary, based on our systematic analyses of NRPs and PLXNs in terms of their expression, association with patient survival, immune subtype, and tumor infiltration, we found that NRP1, NRP2, PLXNA1, PLXNA3, PLXNB3, PLXNC1, PLXND1 were mainly associated with poor prognosis. All these genes, except NRP1, had overall upregulated expression in primary tumors and further upregulated expression in metastatic tumors, were all associated with at least two of the following features: poor survival, more aggressive immune subtypes, and higher tumor infiltration. Interestingly, among these genes, only NRP1 showed significantly decreased overall expression in cancer tumors and further decreased expression in metastatic tumors. However, NRP1 had the highest expression in one of the most aggressive immune subtypes (C6), and was among the genes that showed the strongest association with level of immune infiltration and stromal cells, as well as significantly associated with immune checkpoint molecules. These results indicate that NRP1 may have increased level in TME and explains why it is associated with poor prognosis. In fact, previous studies showed that the upregulation of NRPs often correlate with poor patient prognosis ,84,85,86https://xenabrowser.net/datapages/). The RNA-Seq data was the version that was last updated on 29 December 2016. The tumor samples in TCGA were surgical resection samples obtained from primary tumors that received no prior neoadjuvant treatment. For inter-tumor/pan-tumor analyses, gene expression was normalized to TBP (TATA-box binding protein). The TCGA pan-cancer data include thirty three (33) cancer types, and they are ACC, BLCA, BRCA,CCA, CESC, COAD, DLBC, esophageal carcinoma (ESCA), GBM, HNSC, kidney chromophobe (KICH), KIRC, KIRP, LAML, LGG, LIHC, LUAD, LUSC, MESO, OV, PAAD, PCPG, PRAD, rectum adenocarcinoma (READ), SARC, SKCM, STAD, TGCT, thyroid carcinoma (THCA), THYM, UCEC, UCS, UVM , clinical data, stemness scores based on mRNA (RNAss) and DNA-methylation (DNAss), and immune subtypes were downloaded from xena browser . Gene expression levels and z scores for cell sensitivity data (GI50) for 262 FDA approved drugs or drugs on clinical trials were retrieved for 59 cell lines.In order to investigate the association between gene expression and drug treatment response in cancer cells, the NCI-60 database, which contains data on 60 different cancer cell lines from nine different types of tumors, was accessed using the CellMiner interface (http://bioinformatics.mdanderson.org/estimate/) [The ESTIMATE immune score and stromal score were used to analyze the infiltration levels of immune cells and stromal cells in different tumors . The esttimate/) . Six immtimate/) . Tumor stimate/) .p-value cutoff for significance. For example, we used α = 1/(18 × 17) = 0.003 as cutoff for comparing NRP and PLXN gene expression in tumors compared to adjacent normal tissue as there were 306 tests (18 genes × 17 cancer types). For the survival study, we used α = 0.05 as cutoff without adjusting for multiple comparisons in order for the data interpretation to be consistent with the display of the results in the forest plots.Comparisons of gene expression in all the tumors, all the adjacent paired normal, and all the metastatic tumors across all cancer types, as well as between the normal and tumors in the 17 cancer types which had more than 5 associated adjacent normal samples, were performed using linear mixed effects models. Boxplots were used to show the gene expression across cancer types and a heatmap was used to display the differential expression between tumor and normal in the tested cancer types. Univariate Cox proportional hazard regression models were used to test the association between gene expression and patient overall survival and progression free interval. Spearman or Pearson correlation was used to test the correlation between gene expression at mRNA and protein level, between gene expression and key transducers, stemness scores, stromal score, immune score, estimate score, PD1/PDL1 and CTLA-4 expression, and drug sensitivity score. Analysis of variance (ANOVA) was used to test the association between gene expression and immune infiltrate subtypes and cancer subtypes. All tests were performed using SAS9.4 . Plots were created using R (R Core Team) with packages ggplot2, pheatmap, corrplot, or survminer where appropriate . We usedIn the present study, we are the first to provide a systematic pan-cancer analysis of neuropilins and plexins, which are the primary receptors for the secreted proteins of class-3 semaphorin family. Overall, our study demonstrated that NRP1, NRP2, PLXNA1, PLXNA3, PLXNB3, PLXNC1, and PLXND1 were mainly associated with more aggressive phenotype of cancers and may mainly play tumor promoter roles during cancer tumorigenesis and metastasis, while the rest of the PLXN members PLXNA2, PLXNA4, PLXNB1, and PLXNB2 were more associated with better prognosis and may primarily function as tumor suppressors. Therefore, our study further confirmed their identification as potential therapeutic targets as previously reported, but in a more comprehensive point of view rather than in a limited number of cancer types. However, similar to SEMA3s, to be claimed as a specific tumor promoter or tumor suppressor for each individual member of NRPs and PLXNs, they need to be considered in a cancer type-, tumor subtype-, and immune subtype-specific manner. In summary, even though our conclusions were mainly based on the results from association studies, our work will greatly help future laboratory studies to uncover and validate their roles in tumorigenesis and drug responses, which is important for the development of personalized medicine for cancer treatment and/or to overcome drug-induced resistance.


Atopic dermatitis (AD) is a chronic inflammatory skin disease in humans. In this study, we evaluated the effects of a mixture (NCM 1921) of omega-3 butter, omega-3 beef tallow oil, omega-3 lard oil, caprylic acid, lauric acid, choline, and Fe on AD in 1-chloro-2,4-dinitrobenzene (DNCB)-treated NC/Nga mice. NCM 1921 significantly ameliorated the macroscopic and microscopic signs and reduced skin thickness and mast cell incorporation in the skin lesions of mice with DNCB-induced AD. Furthermore, it reduced serum immunoglobulin E levels; reduced the number of IgE-producing B cells, peripheral blood mononuclear cells, white blood cells, and differential white blood cells; and increased the number of lymphocytes. NCM 1921 normalized the total cell number in dorsal skin tissue, the axillary lymph node, and spleen following DNCB exposure and reduced the number of CD23+/B220+ cells in the axillary lymph node and CD3+ cells in dorsal skin tissue. Moreover, it reduced the levels of interleukin (IL)-4 and IL-13 but increased the levels of interferon-γ in anti-CD3–stimulated splenocytes. Immunohistofluorescence staining showed that NCM 1921 treatment significantly increased claudin1, filaggrin, and Sirt1 protein expressions in AD skin lesions. These results suggest that NCM 1921 could be a valuable remedy for the treatment of AD. Atopic dermatitis (AD) is a chronic inflammatory disease of the skin. It is characterized by eczematous pruritic lesions, elevated levels of allergen-specific immunoglobulin (Ig) E antibodies in the blood, and infiltration of inflammatory cells that produce proallergic cytokines, such as interleukin (IL)-4, IL-15, and IL-13 . The worSeveral previous studies have reported the anti-allergic effects of oils and fatty acids from various sources. Fish oil contains omega-3 polyunsaturated fatty acids (n-3 PUFAs), such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), and can reduce sensitization to allergens; alleviate the severity of AD, eczema, and asthma; and inhibit the expression of IL-1, IL-4, and IL-13 in serum ,9,10,11.w/w)) and mixing. Omega-3 beef tallow oil and omega-3 lard oil were extracted from omega-3 beef tallow and omega-3 lard from omega-3–fed cows and pork , respectively, by supercritical extraction. Caprylic acid was extracted from pinecones of Pinus koraiensis Siebold and Zucc  at temperatures of 65–95 °C under high pressure for 2 days by using supercritical extraction. Lauric acid from coconut oil was purchased from Chemerz Technologies Inc. (the Philippines), and choline and Fe were purchased from Kompass International SA . The composition of NCM 1921, a combination of omega-3 beef tallow oil, omega-3 lard oil, caprylic acid, lauric acid, choline, and Fe, was as follows: omega-3 butter , omega-3 beef tallow oil , omega-3 lard oil (18%. w/w), caprylic acid , lauric acid , choline , and Fe . To prepare omega-3 butter, fat extracted from omega-3 milk  was sterilized at 75 °C for 5 min, and then stored at 4 °C for 24 h after the addition of salt  prepared with olive oil and acetone solution (1:3) once a week. The mice with DCNB-induced AD were divided into the following three groups (n = 8–10): DCNB_ control (DCNB_CTL) group, DCNB_ dexamethasone 3 mg/kg treatment (DCNB_Dexa_3 mg/kg) group, and DCNB_NCM 1921 treatment (DCNB_NCM 1921_300 mg/kg) group. The NC/Nga normal control (NC/Nga_Nor) group was treated with only vehicle (3:1 mixture of acetone and olive oil) without DNCB. The NC/Nga_Nor and DCNB_CTL groups received orally, a vehicle ) with the chow diet. The DCNB_Dexa_3 mg/kg group orally received commercially available dexamethasone 3 mg/kg/day with the chow diet, and the DNCB_NCM 1921 group was given NCM 1921  in the chow diet daily, as described above, for 5 weeks. The doses used were representative of the nontoxic and effective range of NCM 1921 based on previous reports about individual components of NCM1921  [Male NC/Nga mice (7 weeks of age) were purchased from Central Lab Animal Inc. . The study protocol was approved by Daejon University’s Animal Care and Use Committee DJUARB2018-041), and animal studies were performed in accordance with the established institutional guidelines regarding animal care and use and the Guide for Care and Use of Laboratory Animals . The mice were allowed to acclimatize to their environment for 1 week. After 1 week (8 weeks of age), AD was induced by topical application of 0.2% -041, andThe ears of the mice were biopsied, embedded in paraffin wax, and cut to a thickness of 4 μm. The histopathological analysis of the lesions for determining the infiltration of inflammatory cells, such as mast cells, was performed by hematoxylin and eosin (H&E) staining and toluidine blue staining.® 3200 analyzer .To analyze white blood cells , blood was collected from the mice by cardiac puncture. The total cell numbers were counted using a CELL-DYNg, 5 min), primary spleen cells were added to ACK lysis buffer  to remove red blood cells by incubation for 5 min; that was followed by washing twice with RPMI-1640 medium containing 10% fetal bovine serum. The isolated primary ALNs and spleen cells were resuspended in RPMI-1640 medium containing 10% fetal bovine serum and used for the experiment. Briefly, the dorsal skin was removed from the mice, minced using scalpels, and then incubated in PBS containing 1 mg/mL collagenase IV  at 37 °C. After incubation for 40 min, each tissue sample was vigorously pipetted up and down to further dissociate the remaining tissue clumps. The cell suspension was filtered using a 70-mm-pore nylon cell strainer  and then centrifuged for 20 min at 450× g. The cell pellet was collected, and the cells were washed twice. Total cell number was determined using a hemocytometer chamber . The cells obtained were stained immediately with various antibodies for flow cytometry analysis.Axillary lymph nodes (ALNs) and the spleen were isolated from the mice, crushed, and filtered using a 70 μm cell strainer. After centrifugation   for 24 h. After incubation, the supernatants were collected to determine the levels of IL-4, IL-5, IL-13, and IFN-γ.v/v fetal bovine serum and 0.01% w/v sodium azide) for 30 min on ice. The cells were then analyzed using a fluorescence-activated cell sorting analyzer with Cell-Quest software (BD Biosciences).The minced dorsal skin of mice was incubated in PBS containing 1 mg/mL collagenase IV and 2 mg/mL dispase for 40 min at 37 °C. Later, the cells were stained with anti-CD3, anti-CD4, anti-CD8, anti-CD19, anti-CD23, anti-CD69, anti-B220, anti-CCR3, and anti-CD11b antibodies  in staining buffer  in the supernatant of cultured splenocytes, commercially available ELISA kits were used according to the manufacturer’s protocols.p < 0.05 was considered significant.Data are presented as the means ± standard errors of the means (SEMs) and are representative of three independent experiments. One-way analysis of variance (ANOVA) and Duncan’s test were applied using Prism 7.0 ; Since AD lesions cause pruritus and itching, we determined the effects of orally administered NCM 1921 (300 mg/kg) on AD skin lesions. There was no difference in body weight among the treated groups A. As sho+/B220+ cells in PBMCs -α, and IL-2. Th2 lymphocytes secrete IL-4, IL-5, IL-10, and IL-13, which contribute to the pathology of AD. We investigated whether NCM 1921 may regulate the cytokines secreted by of Th1/Th2 cells in the AD model. As shown in Recently, it was reported that abnormal functioning of skin barriers contributes to the pathogenesis of AD. Sirt1 promotes the expression of filaggrin, a skin barrier protein which regulates skin barrier functioning through changes in skin hydration and skin pH ,24,25. CAD is a chronic inflammatory disease of the skin. For typical therapeutic trials to moderate AD, corticosteroids such as dexamethasone are used in the treatment of many conditions, including skin diseases and severe allergies, but, the long-term use of corticosteroids may result in side effects. For this reason, an alternative is necessary for AD patients . In thisAD may occur together with many diseases, such as eczema and allergic conjunctivitis; its symptoms include lichenification, pruritus, epidermal hyperplasia, and tissue remodeling . In our The infiltration of white blood cells increases in AD eczematous lesions. This is particularly due to mast cell infiltration into the diseased site, which leads to increased production of IgE and subsequent pruritus and allergic manifestations. Besides mast cells, neutrophils are involved in allergic skin inflammation. Neutrophils produce leukotriene B4 (LTB4) upon skin scratching, which is essential for their accumulation in mechanically injured skin, for the recruitment of CD4+ T cells to the sites of antigen exposure, and for the development of allergic skin inflammation . In our IgE is an important component of allergic diseases and binds to mast cells, which leads to the secretion of various allergic mediators, such as histamine and cytokines . TherefoAD is generally characterized by increased numbers of infiltrating T cells and activated CD4+ or CD8+ T cells in the dermis. These lymphocytes are involved in the promotion of cell-mediated and humoral immunity through the secretion of various cytokines . The proSkin barrier dysfunction is considered a primary event in AD pathogenesis. Claudin 1 is a transmembrane protein present in the tight junctions of the epidermis, which is an essential component for skin barrier function, such as transepidermal water loss. According to a recent study, reduced claudin 1 protein expression contributes to impaired skin barrier function in lesioned skin of AD patients and these changes are triggered by inflammation ,26,27. S+/B220+ cells, in addition to reducing serum levels of IgE and Th2 cytokines in mice with AD. It also improved skin barrier functioning. Collectively, the findings of the present study suggest that NCM 1921 significantly ameliorates DCNB-induced AD. Therefore, NCM 1921 could be useful in treating skin disorders, including AD, which is accompanied by marked suppressive activity on all the basic components involved in allergy and AD responses.NCM 1921 improved AD symptoms such as itching and pruritus. In addition, it reduced the numbers of mast cells, neutrophils, and IgE-producing CD3


Objective: To provide an overview of what is known about the impact of COVID-19 on weight and weight-related behaviors. Methods: Systematic scoping review using the Arksey and O’Malley methodology. Results: A total of 19 out of 396 articles were included. All studies were conducted using online self-report surveys. The average age of respondents ranged from 19 to 47 years old, comprised of more females. Almost one-half and one-fifth of the respondents gained and lost weight during the COVID-19 pandemic, respectively. Among articles that examined weight, diet and physical activity changes concurrently, weight gain was reported alongside a 36.3% to 59.6% increase in total food consumption and a 67.4% to 61.4% decrease in physical activities. Weight gain predictors included female sex, middle-age, increased appetite, snacking after dinner, less physical exercise, sedentary behaviors of ≥6 h/day, low water consumption and less sleep at night. Included articles did not illustrate significant associations between alcohol consumption, screen time, education, place of living and employment status, although sedentary behaviors, including screen time, did increase significantly. Conclusions: Examining behavioral differences alone is insufficient in predicting weight status. Future research could examine differences in personality and coping mechanisms to design more personalized and effective weight management interventions. Since the COVID-19 pandemic emerged about a year ago, it has infected more than 72 million people and claimed above 1.5 million lives , others Due to the novelty of this disease, the range, nature and magnitude of its impact on weight management in healthy adults remain unclear. Existing systematic reviews tend to focus on the outcomes of patients with obesity diagnosed with COVID-19, but the authors could not find systematic reviews on the effects of COVID-19 on weight and weight-related behaviors ,11. TherThis systematic scoping review was conducted according to the five-phased methodology developed by Arksey and O’Malley . ScopingPhase 1: Research questionsThis study’s research question was developed based on the population, intervention, comparison and outcome (PICO) framework to identify changes in weight and weight-related behaviors during the COVID-19 pandemic in healthy adult populations. Thus, the research question of this study was, “what is known about the changes in weight and weight-related behaviors in healthy adult populations during the COVID-19 pandemic?” Due to the limited number of studies that reports the impact of the COVID-19 pandemic on weight and weight-related behaviors, we included studies that examined populations with a majority of adults  and excluded studies that reported exclusively on populations that were <18 years old. Studies on community-dwelling populations without diseases except being overweight or with obesity during this pandemic were included.Phase 2: Literature searchA systematic three-step search strategy was used to identify relevant literature that was published up to 8 October 2020. First, search terms were generated iteratively through searches on CINAHL and PubMed using the keywords “weight”, “obesity,” and “COVID-19”. MeSH terms were also identified and used as search terms. Second, seven databases  were searched for relevant articles published from the inception of the COVID-19 pandemic to 8 October 2020. The search terms used were “obes*”, “overweight”, “weight”, “COVID”, “COVID-19”, “SARS-COV2”, “SARS-CoV-2”, “2019-nCoV”, “2019 coronavirus”, “behavio*”. More information on the different combinations of the search terms used according to different databases is shown in Phase 3: Study selectionStudies were included if they: (1) described the changes in weight or weight-related behaviors  during the COVID-19 pandemic and (2) were on community-dwelling adults without mention of other diseases except for obesity and being overweight. Studies were excluded if they focused on: (1) biological changes due to a COVID-19 infection; (2) obesity as a risk factor of COVID-19 infections and outcomes; (3) did not discuss weight-related changes related to COVID-19; and (4) were non-primary studies, e.g., simulation/modeling studies.A total of 396 articles were retrieved. After removing 144 duplicate articles, the remaining titles and abstracts were screened for eligibility, which 77 articles were eligible for full-text screening. After excluding articles with reasons shown in Phase 4: Data chartingA data extraction form was created by HSJC and pilot tested on 5 studies. While doing so, common weight-related changes were identified, namely change in dietary behaviors, physical activity behaviors and other lifestyle behaviors. Therefore, the data extraction form was modified to expand the heading “weight-related changes” to the specific ones mentioned earlier. An excel spreadsheet was created to consolidate the extracted data according to the following headings—authors, year of publication, country of origin, study design, survey type, recruitment period, aim of study, follow-up, total number of participants, age, race, baseline BMI, BMI categories, BMI categories’ cutoff scores, proportion of participants overweight, weight change, weight measurement instruments, diet change, diet measurement instruments, physical activity change, physical activity instruments, other weight-related lifestyle behavior changes, predictors of weight, diet, physical exercise and other weight-related lifestyle behavior changes, the significance of change  and important results. Countries of origin were recoded into World Health Organization (WHO) regions, and articles were regrouped into those that evaluated changes in weight, diet and physical activities.Phase 5: Collating, summarizing and reporting the results2 to 27.7 kg/m2; nine reported the proportion of participants who were overweight at baseline (25–60%), and only five articles reported cutoff score used to classify one’s BMI as overweight . More information on the study characteristics is detailed in The 19 included articles represented 61,764 respondents, where the sample sizes of the articles ranged from 90–13,515 (median = 1844), mean/median age ranged from 19 to 47 years old with a median of 33.7 years old. 52.6% of the articles were from the European region , 83.9% were cross-sectional descriptive studies, and all outcomes were collected through online self-report surveys . The majority of the studies recruited participants during the months of April and May (72.2%) and comprised of more females than males . Ten articles reported the participants’ mean baseline BMI that ranged from 20.7 kg/mOut of the 19 articles included in this scoping review ,28,29,30Eleven out of 19 articles mentioned changes in weight where ten articles mentioned weight gain that ranged from 12.8% to 48.6% and six articles mentioned weight loss that ranged from 13.9–19.4%  2,3,4,5,26,28,29Eleven articles described changes in dietary behaviors in terms of appetite  , total cBeing female was a significant predictor of increased appetite , increasFourteen studies reported changes in physical activity, of which four studies reported on the changes in sedentary behaviors during the COVID-19 pandemic . PhysicaPredictors of physical activity included being overweight (OR = 1.8), daily alcohol consumption OR = 4.77), decreased vegetable consumption OR = 3.32), perception of weight increase (OR-2.01), perception of having a healthy diet (OR = 2.11), eating more (OR = 1.87), sedentary for ≥6 h daily (OR = 2.12), exercise ≤30 min a day (OR = 1.99) .77, decr, BMI, ag, perceptNine studies examined changes in other lifestyle behaviors during the COVID-19 pandemic. Six studies reported an increase in sleep hours (49% to 49.9%) ,23,28,30Our scoping review found only 19 studies that examined COVID-19 related changes in weight and weight-related behaviors with mixed conclusions ,28,29,30From reviewing the included studies, up to approximately half of the respondents perceived weight gain during the COVID-19 pandemic period, while up to a fifth of the respondents had reportedly lost weight. There were also inconsistencies in the changes in dietary habits in terms of the consumption of healthy or junk foods and that of physical activity in terms of frequency, duration and energy expenditure. This could be associated with various individual characteristics and prepotent lifestyle habits that influenced weight-related lifestyle changes during the pandemic period. Respondents with a higher baseline BMI was shown to be more likely to experience weight gain, possibly due to a predisposition to eating in response to visual and olfactory food temptations, stress and emotional eating, as mentioned in the results section ,22,26,29Among the studies that examined changes in weight, diet and physical activity concurrently ,4,5,24, Studies included in this review did not illustrate significant associations between weight gain and factors such as alcohol consumption, screen time, education, place of living and employment status, although sedentary behaviors and screen time did increase significantly ,28,29,30Our attempt at identifying the impact of COVID-19 on weight and weight-related behaviors was challenging because while some studies reported the statistical significance of the changes before and after the COVID-19 pandemic, others merely mentioned changes in proportion. Therefore, some changes could have been exaggerated or confounded by other variables such as seasonal changes in temperate countries that cause weight change. Moreover, the time period by which the changes occurred was unclear. It is possible that there exists a behavior change trajectory in coping with the pandemic, where such changes could normalize back to baseline once an individual gets used to the current circumstances—resulting in a minimal net weight change. However, such observation requires a longitudinal study design, which was only used in two studies that reported weight gain in 28.4% to 40% of the respondents ,19. AnotWhile existing studies suggested a higher proportion of people, who gained as compared to those who lost weight, findings regarding the predictors of diet and physical activity changes remain mixed. Moreover, none of the included studies examined other influencing factors of weight-related behaviors, such as personality factors, which could be strong determinants of weight change. Future research could focus on the predictors of different weight-related adaptations  and use more objective outcome measures to enhance the development and accuracy of predictive models for weight management interventions. Health promotion initiatives could also consider exploring the respondents’ needs and preferences in designing weight management programs instead of just prescribing recommendations to follow. Nonetheless, these findings highlighted two behavioral health adaptations—an increase and decrease in the adoption of a healthier lifestyle—to cope with the pandemic measures. This could inform further research, practice and policies in enhancing healthy coping behaviors in a post-COVID-19 era of new norms.


Pklr and Acyl expression. It has recently been demonstrated that ChREBP plays a role in the conversion of gut microbiota-derived acetate to acetyl CoA by activating its target gene, Acss2, in the liver. ChREBP regulates fatty acid synthesis, elongation, and desaturation by inducing Acc1 and Fasn, elongation of long-chain fatty acids family member 6 (encoded by Elovl6), and Scd1 expression, respectively. ChREBP also regulates the formation of very low-density lipoprotein by inducing the expression of Mtp. Furthermore, it plays a crucial role in peripheral lipid metabolism by inducing Fgf21 expression, as well as that of Angptl3 and Angptl8, which are known to reduce peripheral lipoprotein lipase activity. In addition, ChREBP is involved in the production of palmitic-acid-5-hydroxystearic-acid, which increases insulin sensitivity in adipose tissue. Curiously, ChREBP is indirectly involved in fatty acid β-oxidation and subsequent ketogenesis. Thus, ChREBP regulates whole-body lipid metabolism by controlling the transcription of lipogenic enzymes and liver-derived cytokines.Carbohydrate response element-binding protein (ChREBP) plays an important role in the development of type 2 diabetes, dyslipidemia, and non-alcoholic fatty liver disease, as well as tumorigenesis. ChREBP is highly expressed in lipogenic organs, such as liver, intestine, and adipose tissue, in which it regulates the production of acetyl CoA from glucose by inducing  Dietary carbohydrate is metabolized to acetyl CoA, which is a key intermediate in lipid metabolism (Pklr), which is activated by ChREBP and converts phosphoenolpyruvate to pyruvate (Acly) (Acc1) and fatty acid synthase (encoded by Fasn).Excess carbohydrate intake causes hepatic triglyceride accumulation through the activation of carbohydrate response element binding protein (ChREBP) and tabolism . Acetyl tabolism . In the tabolism , 2. In ctabolism , 2. Acettabolism Figure 1Pklr  plays a pivotal role in the pathogenesis of metabolic diseases and tumors –6. ChREBand Fasn , 8, and ic genes –12. Studtabolism –16. Intetl genes , 17–23. tl genes –28. In amulation . ChREBP mulation –32. ThisAcly is expressed mainly in the liver and white adipose tissue and its expression is upregulated at the transcriptional level by glucose-activated ChREBP and the insulin-induced transcription factor sterol response element-binding protein-1c (SREBP-1c)  , 33, 34.REBP-1c) , 36. In REBP-1c) –39  and Elovl6, both of which are required for the synthesis of monounsaturated fatty acids (MUFAs), while SREBP-1c induces Elovl2, fatty acid desaturase 1, and fatty acid desaturase 2, which are required for the synthesis of polyunsaturated fatty acids (PUFAs), as well as Scd-1 and Elovl6 (Chrebp overexpression reduces the concentrations of saturated fatty acids (SFAs), such as palmitic acid (C16:0) and stearic acid (C18:0); and increases that of the MUFA oleic acid (C18:1 n-9)  particles to be delivered to the periphery . These petyl CoA , 34, 51.d Elovl6 Figure 2d Elovl6 –56. Cons8:1 n-9) , 57. Fur8:1 n-9) . Importa8:1 n-9) , 60. Mor8:1 n-9) . Thus, bvia regulation of its nucleocytoplasmic translocation  inhibitor -mediated ChREBP O-GlcNacylation, which enhances ChREBP DNA-binding and protein stability by reducing ubiquitin-mediated degradation –68. Recehylation . H3K4 mehylation Figure 1Chrebp knockout mice -triglyceride, as well as the number of VLDL particles, are lower in out mice , 58, 76.out mice . Furtherinvolved . Hepatictrations . The heprol mice . Becauseycerides , it has ntration , 32, 57.tabolism Figure 3Chrebp knockout mice have lower PAHSA concentrations in their adipose depots  are a recently discovered class of endogenous lipids that have anti-diabetic and anti-inflammatory properties –83. FAHFe depots . RecentlChrebp overexpression reduces plasma OHBA concentration and increases free fatty acid concentrations  or acetoacetyl CoA through ketogenesis. Although ChREBP is activated by feeding, it is also involved in β-oxidation and ketogenesis in the fasting state , 32, 76,ferase-1 . Hepatic oxidase . Therefoogenesis , 58, 76.f21 gene . Althougf21 gene , these rvia acetyl-CoA produced by the conventional pathway (glycolysis and fructolysis) and a novel pathway (conversion of gut microbiota-derived acetate). It also regulates lipoprotein metabolism in the liver and PAHSA production in adipose tissue. Furthermore, ChREBP regulates β-oxidation and ketogenesis in concert with PPARα. However, despite remarkable progress in understanding of the roles of ChREBP in these processes, a number of questions remain regarding its relationship with lipid metabolism. First, to what extent does gut microbiota-derived acetate induce hepatic triglyceride accumulation? Although ChREBP regulates Acss2 mRNA, does acetate itself affect ChREBP transcriptional activity? Second, the role of ChREBP in cholesterol metabolism remains to be determined. Chrebp knockout mice have lower plasma concentrations of cholesterol, despite normal cholesterol synthesis in their livers, and the low plasma cholesterol concentrations may affect physiological processes, such as steroidogenesis in the adrenal gland. Third, the physiological and pathophysiological roles of ChREBP in the fasting state require more in-depth investigation. Because ChREBP is normally activated by glucose, it is conceivable that it regulates post-prandial lipid metabolism. However, it remains unclear why the ability of ChREBP to regulate β-oxidation and ketogenesis evolved. Finally, it remains unclear whether ChREBP suppression modifies the microbiota and lipid metabolism in humans. As discussed above, Chrebp knockout mice have an altered gut microbiota due to impaired fructose absorption from the gut  have made a substantial, direct intellectual contribution to the work, and approved it for publication.This work was supported by grants from Japan Society for the Promotion of Sciences (JSPS) [KAKENHI Grant 17K00850 and 20K11645 (to K. I.) and 17K09825 (to D.Y.)].The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.