Mycobacterium tuberculosis (Mtb). Before the COVID-19 epidemic, TB had the highest mortality rate of any single infectious disease. Vaccination is considered one of the most effective strategies for controlling TB. Despite the limitations of the Bacille Calmette-Guérin (BCG) vaccine in terms of protection against TB among adults, it is currently the only licensed TB vaccine. Recently, with the evolution of bioinformatics and structural biology techniques to screen and optimize protective antigens of Mtb, the tremendous potential of protein subunit vaccines is being exploited. Multistage subunit vaccines obtained by fusing immunodominant antigens from different stages of TB infection are being used both to prevent and to treat TB. Additionally, the development of novel adjuvants is compensating for weaknesses of immunogenicity, which is conducive to the flourishing of subunit vaccines. With advances in the development of animal models, preclinical vaccine protection assessments are becoming increasingly accurate. This review summarizes progress in the research of protein subunit TB vaccines during the past decades to facilitate the further optimization of protein subunit vaccines that may eradicate TB.Tuberculosis (TB), also known as the “White Plague”, is caused by  Mtb is highly contagious and colonizes the respiratory tract through airborne droplets (Tuberculosis (TB), caused by droplets . TB remadroplets . In addidroplets , the lacdroplets , immune droplets .The development of the Bacille Calmette- Guérin (BCG) vaccine was a major milestone in the history of global TB control. Even though it has been more than 100 years since BCG was developed, BCG is still the only licensed TB vaccine worldwide. However, its protective efficiency is still controversial due to its limited immune protection in adults . TherefoThe main TB vaccine candidates include live attenuated vaccines, inactivated vaccines, recombinant viral vector vaccines, and protein subunit vaccines. Since both whole-cell-based and virus-based vaccines pose potential risks to human health, protein-subunit vaccines consisting of protective antigens may be safer and more attractive . HoweverIn this review, we focus on the advances in antigen optimization, adjuvant selection, clinical trials, animal models, and vaccination strategies of protein subunit vaccines, which may foretell the future of TB vaccine research and development.2Mtb a major challenge. Several methods have been used to predict the optimal epitopes for vaccine design.The complex genetic composition, multiple immune evasion strategies, and the lack of rigorous immune markers make the identification of key protective epitopes against + T cells are necessary to protect against TB, they may not be sufficient to obtain a completely protective immune response   molecules (Although CD4response . Many ren (LTBI) . Addition (LTBI) . Therefoolecules .The structure of an antigen determines the specificity, affinity, and accessibility of the binding sites to MHC or antibody, which affects the potency of the immune response . TherefoCombining bioinformatics, structural information, and the AlphaFold2 prediction model to obtain the structural basis underlying protective immune responses to key epitopes is now a popular design strategy to get efficient, long-term, and broad-spectrum responses with multi-epitope TB protein subunit vaccine candidates –17.3Mtb is complex, and many components exhibit immunogenicity. According to different characteristics and associated growth states, Mtb antigens are mainly divided into the following types:The composition of 3.1Mtb contain a large number of glycolipids, lipoproteins, and glycoproteins such as cord factor, phthiocerol dimycocerosates, phosphatidylethanolamine, diacyl trehaloses, lipoarabinomannan, phosphatidyl-myoinositol mannosides, and heparin-binding adhesin, etc.  are involved in the resuscitation and reactivation of dormant ith LTBI . There anfection .3.5Mtb, but 16 genomic region of difference (RD) antigens are deficient in BCG compared to Mtb  and glycolipid immunomodulator trehalose-6,6-dibehenate (TDB). DDA is a potent adjuvant capable of eliciting cellular and humoral immune responses . TDB is 4.2Quillaja saponaria extract), co-prepared in the presence of cholesterol  and the saponin QS-21 (lesterol . MPL actresponse .4.3GLA-SE is a mixture of the TLR4 agonist glucopyranosyl lipid A (GLA) and squalene emulsion (SE) . GLA is 4.4IC31 comprises the synthetic, positively charged antimicrobial peptide KLKL5KLK and oligodeoxynucleotide 1a (ODN1a) . ODN1a i4.5+ T cell responses (Dextran/CpG is a novel adjuvant developed with diethyl aminoethyl (DEAE)-dextran and CpG ODN . CpG is esponses . Dextranesponses , 83.4.6Advax is a novel cationic adjuvant based on the Delta inulin isoform and has a diameter of about 1-2 μm . Advax-b4.73) 3) . CpG ten3) . BC02 in3) .4.8DDA/poly(I:C) is composed of cationic liposome vector DDA and polyriboinosinic acid–polyribocytidylic acid, poly(I:C). Poly(I:C) mimics viral dsRNA and is a promising immune stimulator candidate for vaccines against intracellular pathogens. Poly(I:C) signaling primarily depends on TLR3 and melanoma differentiation-associated gene-5 (MDA-5) . Moreove4.9With the development of nanotechnology and the increasing understanding of immune responses to metals, different types of inorganic nanoadjuvants have been developed, including manganese , iron 8, silicon5Pre-clinical and clinical trials are always needed to evaluate the safety and efficacy of novel vaccine candidates. We summarize the significant progression of protein subunit vaccines in recent trials.5.15.1.1Mtb that is up-regulated during LTBI  as an adjuvant  rather than effector memory T cells  (LT70 is a multistage protein subunit vaccine composed of antigens prominent at different metabolic stages of the adjuvant . In a mu of mice . Another of mice . In addiapacity) .5.1.3+ or IL-2+ CD4+ T cells and IFN-γ+ CD8+ TEM cells in spleen more effectively than BCG  administered subcutaneously adjuvanted with DMT , 44. CMFthan BCG –45. CMFOTBI mice , suggest5.1.4Mtb–specific antigens  with a protective effect against Mtb pulmonary infection , H74 , and H107  are protein subunit vaccines composed of antigens , 50. H64nfection . H107 va5.25.2.1AEC/BC02 is a vaccine candidate for LTBI consisting of Ag85B and the fusion protein ESAT-6-CFP10 with adjuvant BC02 . Preclin5.35.3.1Mtb antigens  with GLA-SE as an adjuvant (Mtb virulent strain H37Rv and multidrug-resistant strain TN5904 (Mtb-infected mice and nonhuman primate (NHP) models   . In a phα, IL-2) . Phase Iα, IL-2) .5.3.3E is composed of two immunogenic Mtb fusion proteins (Mtb32A and Mtb39A) with AS01E as an adjuvant. M72/AS01E protected against Mtb invasion after aerosol infection when administered intramuscularly to C57BL/6 mice and guinea pigs , CD8+  T-cells and antigen-dependent NK cell IFN-γ production , CD8+ T cells secreting IFN-γ, and IgG responses  and cynomolgus macaques (CM) are the most commonly used primate models for TB vaccine research. It is well known that there are differences between macaque species in their ability to control disease progression, with RM showing higher rates of progression and higher levels of bacterial burden compared to CM . RM are Mtb infection , but highly susceptible to infection with the closely related Mycobacterium bovis. They can form granulomas, liquefaction, and cavities similar to the events found in humans, making them suitable for the study of processes leading to transmission of Mtb as well as for vaccine and drug research. In addition, the rabbit model has been used in studies of cavitary, spinal, joint, cutaneous, and meningeal TB  and clinical trials (H56 and ID93)  , 63, 71.+ Th1 cells, and the functional plasticity of these cells is limited. Moreover, BCG-generated immunity impedes the subsequent induction of additional protective T cells with memory and lung homing potential by the booster vaccine  may circumvent this dilemma  can elicit more robust protection than BCG when used alone, suggesting that such a vaccine could use as an alternative to BCG , 43, 68.EM cells. A booster regimen with longer intervals of 4 weeks appeared to favor the generation of long-term TCM cells  have great potential for development and utilization. By far, multiple protein subunit TB vaccines have entered clinical or preclinical trials and have broken the barrier that BCG can only be used for pre-infection prevention. And even some vaccines have shown surprising protection in post-exposure prophylaxis in people with LTBI and in the treatment of people with active TB infection. Rapidly evolved bioinformatics and structural informatics technologies represent a large reservoir to filter out plentiful numbers of XF, and ZH contributed to the conception and revised of this manuscript. ZY and JX drafted and revised the manuscript. All authors contributed to the article and approved the submitted version.


This case report presents a 43-year-old female with a history of common variable immunodeficiency (CVID) and a recent diagnosis of mesenteric volvulus. The patient presented with symptoms of partial small bowel obstruction and was diagnosed with obstruction and mesenteric volvulus primarily affecting the proximal jejunum. During the exploratory laparotomy, a probable polyposis syndrome and a possible adenocarcinoma of the small bowel were identified. Pathological examination confirmed the presence of B-cell lymphoma in the proximal jejunum. The patient underwent treatment with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone (RCHOP) chemotherapy and showed improvement in symptoms. The case highlights the increased risk of malignancies, particularly lymphomas, in individuals with CVID and the challenges in diagnosing and treating lymphoid neoplasms in this population. The prevalence of primary immunodeficiencies and immunological dysregulatory diseases is 41.4 or 50.5 per 100,000 people, respectively. According to research conducted in the United States and Europe, common variable immunodeficiency (CVID) is the most prevalent adult humoral immunodeficiency . MalignaA 43-year-old female with a past medical history of CVID and receiving intravenous immunoglobulin of 15 grams once a week has a surgical history that includes a prior cesarean section and tubal ligation presented with complaints of abdominal pain and abdominal distension. The patient had been experiencing chronic diarrhea for four months, which was initially attributed to a diagnosis of Giardia. However, her symptoms worsened over the past 24 to 48 hours, accompanied by sudden abdominal pain and distention. The patient denied nausea, vomiting, significant weight loss, difficulty swallowing, and blood in stools. In terms of vital signs, the patient's blood pressure ranged from 87/45 to 100/55 mmHg, pulse rate ranged from 90 to 97 beats per minute, respiratory rate ranged from 20 to 22 breaths per minute, and weight was recorded at 51.8 kg over a 24-hour period.The patient denied any history of smoking, use of smokeless tobacco, alcohol consumption, or drug use. However, her family history revealed a case of breast cancer in her mother. Laboratory tests showed potassium -3.1 =3.7-5.2 mEq/L), mild anemia , aspartate transaminase -52 U/L (n=8-33 U/L), alanine transaminase -49 U/L (n=4-36 U/L), alkaline phosphatase -190 U/L (20-130 U/L), total protein -4.2 g/dL (n=6-8.3 g/dL), and albumin -2 g/dL (3-4-5.4 g/dL). CA 125 elevated with the level of 40 U/mL (n=0-35 U/mL). Tumor markers, serum AFP, CA 19-9, and CEA, are negative. Stool cultures were negative for Helicobacter pylori, Campylobacter jejuni, Escherichia coli 0157, Salmonella, Shigella, Yersinia, Aeromonas, Plesiomonas, and ova and parasites.The CT scan showed swirling of the mesentery, confirming the presence of mesenteric volvulus and dilated proximal small bowel loops Figure . The patThe surgical report indicated a probable polyposis syndrome and adenocarcinoma of the small bowel. Pathological examination of the resected tissue indicated B-cell lymphoma with Ki-67 close to 90% in the proximal jejunum, along with intramucosal reactive lymphoid follicles. Further classification was requested through FISH studies, which showed no evidence of MYC, BCL2-IGH, or BCL 6 rearrangements. The bone marrow was negative for DLBCL.The patient's hospital course involved postoperative recovery after a partial small bowel obstruction due to mesenteric volvulus. A seven-day course of IV piperacillin and tazobactam was administered for broad-spectrum antibiotic coverage to prevent infection in the postoperative period. The patient showed gradual improvement in her abdominal pain and distention following the surgery.Regarding the treatment for B-cell lymphoma, the patient completed six cycles of rituximab 600 mg, cyclophosphamide 1,155 mg, doxorubicin 77 mg, vincristine 2 mg, and prednisolone oral 100 mg (RCHOP) and repeated her completion PET-CT which showed 1.9 x 0.9 cm dense central mesenteric node again visualized with the maximum standardized uptake value (SUV max) of 4.0 and Deauville score of 4. The previous showed 2.0 x 1.0 cm with an SUV max of 4.1 in the left mesentery. In addition to antibiotic therapy, the patient received intravenous fluids to maintain hydration and electrolyte balance. Pain management was achieved using analgesics, primarily focusing on opioid-sparing medications to minimize the risk of postoperative ileus.While selective IgA deficiency is the most common primary immunodeficiency, it is often asymptomatic. On the other hand, CVID is the most clinically relevant primary immunodeficiency. The well-established definition of CVID includes three essential features: hypogammaglobulinemia affecting two or more immunoglobulin isotypes , recurrent infections of the sinopulmonary system, and impaired functional antibody responses . ResearcThe underlying mechanisms contributing to lymphoma development in CVID remain incompletely understood, but factors such as high IgM levels, late-onset combined immunodeficiency phenotype, and polyclonal lymphoproliferative disease have been associated with an elevated risk . AdditioCVID is a clinically relevant primary immunodeficiency associated with hypogammaglobulinemia and recurrent infections. Individuals with CVID are at an increased risk of developing malignancies, including lymphomas. The mechanisms underlying lymphoma development in CVID are not fully understood. Primary GI lymphomas, particularly B-cell lymphomas, are more prevalent in CVID patients and require careful diagnosis due to overlapping features with lymphoid hyperplasia. Further research is needed to better understand the underlying mechanism and identify specific risk factors that contribute to lymphoma development in CVID patients by focusing on investigating the intricate interplay between immune dysregulation, genetic predisposition, and the microenvironment to optimize management strategies for lymphomas in CVID patients. Prompt recognition and appropriate treatment, such as chemotherapy regimens like RCHOP, are crucial for improving outcomes in CVID patients with lymphomas.


CHEK2 as the first moderate-penetrance TGCT predisposition gene. Here, we analyzed CHEK2 in 129 TGCT cases unselected for age of onset, histology, clinical outcome, and family history of any cancer, and the frequency of identified variants was compared to findings in 27,173 ancestry-matched cancer-free men. We identified four TGCT cases harboring a P/LP variant in CHEK2 , which reached statistical significance , 4.06; 95% CI, 1.59–10.54) as compared to the control group. Cases with P/LP variants in CHEK2 developed TGCT almost 6 years earlier than individuals with CHEK2 wild-type alleles . No association was found between CHEK2 status and further clinical and histopathological characteristics, including histological subtypes, the occurrence of aggressive TGCT, family history of TGCT, and family history of any cancer. In addition, we found significant enrichment for the low-penetrance CHEK2 variant p.Ile157Thr , 3.69; 95% CI, 1.45–9.55). Thus, we provide further independent evidence of CHEK2 being a moderate-penetrance TGCT predisposition gene.Testicular germ cell tumors (TGCTs) represent the most frequent malignancy in young adult men and have one the highest heritability rates among all cancers. A recent multicenter case–control study identified  Testicular germ cell tumor (TGCT) is the most frequent malignancy in young men aged 15–45 years. Based on histology, TGCTs are classically divided into seminoma and non-seminoma, the latter including choriocarcinomas, teratomas, and yolk sac and embryonal carcinomas. Moreover, in about 5% of individuals, germ cell tumors (GCTs) occur at extragonadal sites . This coRegarding the germline genetic basis, TGCTs are actually estimated to have the third highest heritability among all cancers , with anCHEK2, encoding the checkpoint kinase 2 (CHK2), are strongly enriched in all three cohorts, thereby identifying CHEK2 as the first TGCT moderate-penetrance susceptibility gene [CHEK2 were 4–6 times more likely to develop TGCTs than unaffected male individuals and, on average, had a 6-year earlier age of presentation than TGCT men with wild-type CHEK2 alleles. Moreover, the low-penetrance CHEK2 variant (p.Ile157Thr) was found to be a Croatian founder TGCT risk variant [Indeed, only recently, using different discovery strategies in three independent TGCT cohorts, a multicenter study has shown that pathogenic/likely pathogenic (P/LP) germline variants in ity gene . Namely, variant .CHEK2 analysis in 129 men of European ancestry with TGCTs, unselected for early onset or a positive family history of TGCTs, which were ascertained at the University Medical Center Hamburg-Eppendorf.To further validate these findings, we performed a comprehensive A total of 129 men of European ancestry with a primary germ cell tumor, 107 with a testicular germ cell tumor (TGCT) and 22 with an extragonadal germ cell tumor (EGCT) , unselecCHEK2 were performed as previously described [CHEK2 variants followed the American College of Medical Genetics and Genomics (ACMG) criteria, as previously described [The extraction of genomic DNA from whole-blood samples, PCR amplification, and the Sanger sequencing of all 14 coding exons of escribed ,14. In aescribed ,14, the escribed . The cliescribed ,14.t-test with Welch’s correction were performed using GraphPad Prism 8.For statistical analysis, Fischer’s exact test and an Unpaired A detailed clinical and pathological characterization of the 129 germ cell tumor (GCT) men included in the study is shown in Family history of GCT occurrence was available for 115 cases (89%), and only four cases (3.48%) reported a GCT in first- and second-degree relatives. Moreover, family history of any malignancy was available for only 92 cases (71.3%). Out of the latter, 58 cases (63%) reported at least one malignancy in first- and second-degree relatives .CHEK2 followed by PCR amplification for the detection of exon 9_10 deletion identified altogether six heterozygous, non-synonymous variants. Two cases harbored the c.1100delC, p.Thr367Metfs15*, and one case harbored the c.444 + 1G > A (IVS2 + 1G > A). Notably, both of these variants have been previously identified in TGCT cases and classified as pathogenic [http://www.fruitfly.org/seq_tools/splice.html, accessed on 10 May 2023) [CHEK2 germline variants in four GCT cases . Moreover, a comparison to the control group reached statistical significance, and the LOF germline CHEK2 variant carriers were four times more likely to develop TGCTs than unaffected male individuals , 4.06; 95% CI, 1.59–10.54; The direct Sanger sequencing of ay 2023)  suggesteay 2023) ,18. Takep = 0.0259; In addition, we identified four cases  harboring the low-penetrance variant c.470T > C, p.Ile157Thr , which wAdditionally, we identified a single GCT case harboring c.358A > G, p.Ser120Gly, and a single GCT case harboring c.1525C > T, p.Pro509Ser. The p.Ser120Gly variant is located within the forkhead-associated domain (FHA) and has so far not been identified in large datasets, such as the Exome Aggregation Consortium (ExAC)  or the GCHEK2 germline variant status, we next performed a case–case analysis. Carriers of CHEK2 LOF variants developed TGCTs almost 6 years earlier than non-CHEK2 LOF carriers . However, this finding reached only borderline significance (p = 0.2315). Moreover, a further comparison regarding the histology, the occurrence of aggressive TGCTs, family history of TGCTs, and family history of any cancer all revealed statistically insignificant p-values , 4.06; 95% CI, 1.59–10.54; CHEK2 as a moderate-penetrance TGCT predisposition gene. Concerning our patient cohort, the majority presented with metastatic disease and non-seminomatous histology. Furthermore, with 17%, the fraction of extragonadal germ cell tumors is relatively high. This is comprehensible, as it can be assumed that the number of patients with advanced and/or refractory disease is higher in a referral center. However, as the patients detected with CHEK2 germline variants are under current follow-up care after treatment, there is no evidence that this genetic alteration leads to a more aggressive course or chemotherapy refractoriness. To corroborate these findings, we have ascertained a further cohort and carried out the first independent replication study in men with TGCTs of European ancestry who were unselected for age of onset, clinical outcome, family history of TGCTs, and family history of any cancer. Indeed, we identified pathogenic or likely pathogenic (P/LP) loss-of-function (LOF) CHEK2 variants [Several large-scale analyses in various other cancers suggested a reduced penetrance of p.Ile157Thr as compared to the P/LP variants ,23,31. Pvariants  and by avariants . Out of variants ,21, p.Ilvariants . Here, wCHEK2 variant status, suggested that P/LP CHEK2 variant carriers have an earlier age of onset (with a mean of 5.95 years) and an increased risk of developing a contralateral TGCT as compared to CHEK2 wild-type allele carriers [CHEK2 LOF in this study also showed a trend, which did not, however, reach statistical significance, towards an earlier age of onset  polymerase (PARP) inhibitors might in the future be an additional utility for germline CHEK2 genetic testing in TGCT cases, as is already established for metastatic castration-resistant prostate cancer [Last but not least, the sample size of our cohort and, accordingly, the usage of a well-established large ExAC dataset are clear limitations of our study. For this reason, we additionally compared the clinicopathological characteristics of all TGCT terature ,21. Unfognancies ,24, we se cancer .CHEK2 as a moderate-penetrance TGCT susceptibility gene associated with an early age of onset. In addition, our data suggest that the low-penetrance CHEK2 variant, p.Ile157Thr, leads to increased TGCT risk beyond the Croatian population. In addition, we suggest that all men with a TGCT should be offered a germline CHEK2 analysis.Taken together, our study provides further evidence of


Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infects gut luminal cells through the angiotensin-converting enzyme-2 receptor and disrupts the gut microbiome. We investigated whether the gut microbiome in the early stage of SARS-CoV-2 infection was associated with the prognosis of coronavirus disease (COVID-19).Thirty COVID-19 patients and 16 healthy controls were prospectively enrolled. Blood and stool samples and clinical details were collected on days 0 (enrollment), 7, 14, and 28. Participants were categorized into four groups by their clinical course.p=0.003). A high abundance of the genus Dialister , species Peptoniphilus lacrimalis , and Anaerococcus prevotii  was associated with a good prognosis. Starch, sucrose, and galactose metabolism was highly activated in the gut microbiota of the poor prognosis group. Glucose-lowering diets, including whole grains, were positively correlated with a good prognosis.Gut microbiota composition varied during the clinical course of COVID-19 and was closely associated with cytokine levels (Gut microbiota may mediate the prognosis of COVID-19 by regulating cytokine responses and controlling glucose metabolism, which is implicated in the host immune response to SARS-CoV-2. During the early stage of the COVID-19 pandemic, most patients with COVID-19 presented with self-limiting mild respiratory illness, although 14% suffered from severe disease, and 2.45% of these patients died (9 is a novia the angiotensin-converting enzyme 2 receptor (ACE2), which is present throughout the body, including in the respiratory and gastrointestinal tracts  and metabolism. We investigated serial changes in gut microbiota and cytokines up to 4 weeks after symptom onset and analyzed their correlation with the prognosis of COVID-19.22.1In this prospective cohort study, 30 hospitalized patients with COVID-19 and 16 healthy controls were recruited from January 8, 2021, to September 9, 2021, at two tertiary hospitals in the Republic of Korea . SARS-CoV-2 infection was confirmed using real-time (RT) polymerase chain reaction (PCR) of nasopharyngeal specimens. Patients who received probiotics within 4 weeks of symptom onset were excluded. Healthy individuals were enrolled as controls after excluding those who received any medications, including antibiotics, probiotics, laxatives, and motility drugs, that could affect the microbiome within 4 weeks prior to study enrolment. All controls tested negative for SARS-CoV-2 on nasopharyngeal RT-PCR and serological tests. Fecal and blood samples were collected serially on days 0 (enrolment), 7, 14, and 28. Patient data related to demographics, medications, chest radiography findings, laboratory results, and dietary records were obtained by two trained physicians. The analysis period was classified into week 1 (days 0–7), week 2 (days 8–14), week 3 (days 15–21), week 4 (days 22–28), week 5 (days 29–35), and week 6 (days 36–42) in the post-infection period.Patients were assessed at enrolment based on the eight-category National Institute of Allergy and Infectious Disease Ordinal Scale score  and the Institutional Review Board of Chungbuk National University Hospital (2020-12-021). All participants provided informed consent. All procedures were performed according to the ethical standards of the institutional and/or national research committee and in accordance with the Declaration of Helsinki, 1964, and its later amendments or comparable ethical standards.2.2Fecal samples were collected using Faecal Swab DNA Preservation and Transport Kits  containing nucleic acid preservation media. Fecal samples in the fecal swab transport medium were stored at −80°C. Blood samples were obtained by venipuncture and were collected in a serum-separating tube, which was subsequently centrifuged at 2500 rpm at −4°C for 10 min, and the serum-containing supernatant was pipetted into a clean plastic screw-cap vial and stored at −80°C.2.3We selected five cytokines that are known to influence the clinical course of COVID-19 , 7. Bloo2.4® Pro DNA kit  following the manufacturer’s instructions. At ChunLab, Inc. , PCR amplification was performed using fusion primers that target the V3–V4 regions of the 16S rRNA gene, and mixed amplicons were pooled and sequenced using the Illumina MiSeq Sequencing System  according to the manufacturer’s instructions. The microbiological analysis process includes DNA extraction, PCR amplification, and sequencing of 16S rRNA and sequence data processing and analysis, which was performed using EzBioCloud 16S-based Microbiome Taxonomic Profiling and ChunLab’s bioinformatics cloud platform  that was developed as part of the Korea National Health and Nutrition Examination Survey (KNHANES) , 9. The 2.6t-test or Wilcoxon rank-sum test) were used to detect intergroup differences. The Kruskal–Wallis test was used to evaluate differences in the measured values among the four prognostic groups, and multiple comparisons were adjusted using the Bonferroni correction. The Spearman rank test was performed for the 112 food items and nutrient intake for the correlation analysis. Statistical analyses were performed using R version 4.1.2 . Associations between the gut microbiota composition and serum inflammatory cytokine and antibody concentrations of the patients with COVID-19 were analyzed using the VEGAN, ggPLOT2 package in R. Principal component analysis of variance was conducted to identify the best set of variables that describe the community structure by fitting linear models to distance matrices and using a permutation test. Regarding the prognosis-stratified groups, five serum inflammatory cytokines were presented along the direction, and these were scaled by their correlation such that the weak predictors had shorter arrows than the strong predictors. The plotted cytokine arrows are depicted in the microbiome NMDS plot using the ggplot2 graph, which keeps the relative r2-scaled lengths of the arrows using VEGAN in the R package.All continuous variables are expressed as the median ± interquartile range, and categorical variables are presented as numbers (percentages). Non-parametric tests . Regarding the laboratory findings, the lactate dehydrogenase (LDH) level  was lower in group A than in the others.Among the 30 patients with COVID-19, 5 were in group A (recovery from mild COVID-19), 9 were in group B (an improvement from moderate to mild COVID-19), 12 were in group C (an improvement from severe to mild COVID-19), and 4 were in group D (deterioration). As presented in 3.2With regard to the alpha diversity , a significant difference was observed between the control group and each COVID-19 group; however, the alpha diversity of group B in week 1 and group A in week 2 were indistinguishable from that of the control group  and over time (weeks) after hospitalization (p<0.001). For a comprehensive analysis, we analyzed changes in the microbiome of the four groups over time after symptom onset (p=0.218). However, intergroup differences became apparent over time at week 2 (p=0.017). After the 15th day of symptom onset (weeks 3 and 4), the intergroup difference disappeared . The groups with clinical improvement (groups B and C) and the control group showed closer microbial composition at the convalescent stage, whereas in group D, whose clinical course worsened, microbial composition remained far from that of the control group. In group A, the intergroup distance was maintained far from that of the control group despite the clinical improvement, which might be due to the underlying medical conditions.For analyzing beta diversity by clinical course, principal coordinate analysis using Bray–Curtis distances was performed and revealed significant intergroup differences . Furthermore, CCA showed a significant correlation between the gut microbiota and cytokines when analyzed using the Charlson comorbidity index  and the time that had elapsed after symptom onset  (p=0.046) and week 3-6 , whereas anti-S IgG antibody titers were similar. Furthermore, the CCA was conducted for microbiome and inflammatory markers over time (p=0.043) and LDH (p=0.002) levels differed significantly between the groups at week 1-2, whereas CRP (p=0.004), LDH (p=0.037), procalcitonin (p=0.018), and D-dimer (p=0.030) levels significantly differed among the groups at week 3-6.By using canonical correlation analysis (CCA), the gut microbiota composition of 30 patients with COVID-19 was visualized by prognostic groups with five serum cytokines – IFN-γ, IL-2, IL-10, TNF-α, and anti-SARS-CoV-2 S immunoglobulin G (IgG) titers (p=0.014) and 3 (p=0.007). At week 4, the IFN-γ (p=0.022), IL-10 (p=0.010), and IL-6 (p=0.014) levels differed significantly between the prognosis groups. At the early stage of infection (week 1-2), IFN-γ and IL-6 levels were lower in groups C and D than in groups A and B  effect size >2.5 were considered good taxonomic markers Table 2.Peptoniphilus lacrimalis , Anaerococcus prevotii , genus Dialister , Mogbacterium JRNA_g , and Mogibacterium JRNA_g JRNA_s  were good prognosis-related taxonomic markers. However, there were no taxonomic markers that significantly indicated a poor prognosis.We used LDA effect size to identify prognosis-related taxonomic markers during the early period (weeks 1 to 2) after symptom onset. We found that 3.4p=0.015) and K07483  were identified as good functional biomarkers.We investigated functional biomarkers for predicting prognosis similarly to the process followed for taxonomic markers Table 3.p=0.038), K21064 , K02756 , and K03488  were poor functional biomarkers. The PICRUSt algorithm revealed that the KEGG modules M00271 , M00632 , and ko00500  were dominant in the poor prognosis group. Using the MinPath algorithm, the KEGG pathway ko00052  was abundantly detected in the poor prognosis group.In contrast, the KEGG orthologs K01223  was gradually restored, but not to the level of healthy controls until weeks 4-6 post-infection. In comparison, decreased alpha diversity persisted in the poor prognostic group (D). As ACE2, the main receptor of SARS-CoV-2, is highly expressed as a neutral amino acid transporter in the gastrointestinal tract; the virus can invade the gut, disrupting mucosal integrity and microbial dysbiosis , 11. AccBased on clinical findings, response to therapy, and clinical outcomes, COVID-19 can be subdivided into three phases: viral, inflammatory, and recovery , 14. ThuIn this study, serial changes in the gut microbiota composition were related to cytokine responses according to the clinical course of COVID-19. These correlations may be supported by the characteristics of the taxonomic markers that reflected the prognosis of COVID-19 in this study. Propionate, a major short-chain fatty acid, is mainly produced through the succinate decarboxylation pathway that involves the Dialister species . PropionWith regard to the functional biomarkers, we found that the starch, sucrose, and galactose metabolism pathways were highly activated in the poor prognosis group. Our findings show that obesity and diabetes mellitus are risk factors for severe COVID-19 , 29. VirDietary carbohydrate restriction improves glycaemic control, thereby mitigating COVID-19 , 37. We There are some limitations in this study. First, there was a remarkable difference in the ages between healthy controls and COVID patients. Second, the sample size of healthy controls was small. Third, both Alpha and Delta variant viruses circulated during the study periods; Alpha variant was dominant between January and May of 2021, and was gradually replaced by the Delta variant since June. Viral factor might have affected overblown variations in some results . FinallyIn conclusion, this study showed that the gut microbiota and cytokines are closely related. Gut microbiota may influence immune responses and glucose metabolism, which in turn are involved in the prognosis of COVID-19. High starch, sucrose, and galactose metabolism in the gut microbiota during SARS-CoV-2 infection may increase glucose levels, thereby promoting SARS-CoV-2 replication and proinflammatory cytokine production. The gut microbiota is strongly implicated in host immune responses to SARS-CoV-2 and regulates timely cytokine activation. Early-inadequate and late-dysregulated cytokine responses are associated with poor prognosis of COVID-19.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. The datasets generated and/or analyzed during the current study are available in the NCBI repository, BioProject gut microbiome [Accession No. PRJNA941155].The studies involving human participants were reviewed and approved by Institutional Review Board of Korea University Guro Hospital (2020GR0570) Institutional Review Board of Chungbuk National University Hospital (2020-12-021). The patients/participants provided their written informed consent to participate in this study.Conceptualization, HS, JK, JN, HC, WK, and JS. Methodology, HS, JK, Y-HH, EN, HSS, and SL. Formal analysis, HS, Y-HH, and SL. Investigation, HS, JK, HH, and JY. Data curation, HS and SL. Writing - original draft, HS, Y-HH, SL, and JS. Writing–review and editing, HS and JS. Visualization, HS and SL. Supervision, HS, SL, and JS. Funding acquisition, HS, SL, and JS. All authors contributed to the article and approved the submitted version.


Liver cancer, comprising hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA), is a leading cause of cancer-related deaths worldwide. The liver is a primary metabolic organ for progesterone (PRG) and PRG exerts its effects through classic nuclear PRG receptors (nPRs) and non-classic membrane PRG receptors (mPRs) or a combination of both. Previous studies have shown that the CCM signaling complex (CSC) couples both nPRs and mPRs to form the CmPn (CSC-mPR-PRG-nPR) signaling network, which is involved in multiple cellular signaling pathways, including tumorigenesis of various cancers. Despite advances in treatment, 5-year survival rates for liver cancer patients remain low, largely due to the chemoresistant nature of HCCs. The lack of sensitive and specific biomarkers for liver cancer diagnosis and prognosis emphasizes the need for identifying new potential biomarkers. We propose the potential use of CmPn members’ expression data as prognostic biomarkers or biomarker signatures for the major types of hepatic cancer, including HCCs and CCAs, as well as rare subtypes such as undifferentiated pleomorphic sarcoma (UPS) and hepatic angiosarcoma (HAS). In this study, we investigated the CmPn network through RNAseq data and immunofluorescence techniques to measure alterations to key cancer pathways during liver tumorigenesis. Our findings reveal significant differential expression of multiple CmPn members, including CCM1, PAQR7, PGRMC1, and nPRs, in both HCCs and CCAs, highlighting the crucial roles of mPRs, nPRs, and CSC signaling during liver tumorigenesis. These key members of the CmPn network may serve as potential biomarkers for the diagnosis and prognosis of liver cancer subtypes, including rare subtypes. Liver cancer is a significant cause of cancer-related death, ranking third globally, while also being the sixth most commonly diagnosed cancer . The twoAmong the mPRs, mPRα (PAQR5), mPRβ (PAQR6), mPRγ (PAQR7), mPRδ (PAQR8), and mPRε (PAQR9) have been investigated for their involvement in angiogenesis and tumorigenesis ,30. TherIn this study, we examined the mRNA expression of essential CmPn players, including the CSC (CCM1-3), mPRs , and nPRs (PGR1/2), along with the current liver biomarker AFP, across various liver tissue and cancer types, along with patient clinical information. In addition, we utilized immunofluorescence (IF) techniques to analyze protein expression of essential CmPn members, such as CCM1/3, PAQR7/8, PGRMC1, and nPRs. Our results demonstrate that several key members of the CmPn network have the potential to be utilized as biomarkers for diagnosing and prognosing different liver cancer subtypes at both the transcriptional and translational levels, as revealed through our comparative bioinformatics and IF approaches.Clinical profiling for hepatic cancers utilizing the NCI-GDC data portal. Utilizing the Genomic Data Commons (GDC) data portal from the National Cancer Institute (NCI), we assessed all available sociological/clinical/diagnostic data for patients diagnosed with liver  or intrahepatic bile duct cancers  [ma, CCA) ,39,40,41AFP, using microarray expression data.Differential expression profiling of key CmPn members, along with  Expression analysis was performed as previously described [escribed ,17,23,26escribed , for botescribed .AFP, for both HCCs and CCAs, based on histological sample type . We then repeated expression profiling for each cancer type based on immune subtypes, in a pair-wise fashion. Finally, we evaluated expression profiling of key CmPn members, along with AFP, between HCCs and CCAs, to evaluate overlaps and differences in expression between these two cancer subtypes. Significant differential expression was performed using Xenabrowser with a cutoff p-value <0.05. Gene ontology, KEGG, and reactome pathways enrichment analysis was performed from the RNAseq data using Enrichr. Significance was evaluated using a cutoff p-value <0.05 and FDR cutoff value <0.1.Additionally, RNAseq data from the TCGA-TARGET-GTEx, as well as the TCGA-PANCAN databases, were used to investigate differential expression of key CmPn members, along with Deparaffinization of paraffin-embedded tissue sections. IF processing of slides was performed as previously described [escribed ,17,23,26Antigen Retrieval. Tissue sections were washed in 1X PBS 3 times, 3 min per wash, before being permeabilized in PBS containing 0.2% Triton X-100 (wash buffer) for 10 min. Tissue slides were then submerged in 10mM sodium citrate buffer  containing 0.01% Triton X-100. Tissues were maintained at 95–98 °C for 30 min in citrate buffer and then set aside to cool at room temperature (RT).Blocking and Antibody Incubation. Following antigen retrieval, slides were washed 3 times in wash buffer at 3 min per wash at RT. The tissues were blocked using Pierce Fast Blocker Buffer (Fisher) for 90 min at room temperature to optimize the blocking process. A hydrophobic pen was used to draw a barrier around the tissue sections to conserve antibodies. All primary antibodies were diluted in wash buffer and Pierce Fast Blocking Buffer (1:1). An initial 500 uL was added to the tissues and left to incubate for 2 h at RT in the dark. Primary antibody was removed, and tissues were washed 3 times in wash buffer at 3 min per wash at RT. An amount of 400 uL of secondary antibody, which was diluted in wash buffer and Pierce Fast Blocking Buffer (1:1), was added to the slide and left to incubate for 2 h at RT. All antibodies used are listed in Nuclear Staining and Mounting/Sealing. Tissue sections were stained with DAPI by adding two drops of mounting media containing DAPI directly to the sections. Tissues were left to rest overnight at 4 °C in the dark to allow efficient staining of DAPI. Slides were sealed with nail polish the next day and allowed time to dry before imaging.Imaging and Quantification. IF imaging/quantification of microarray slides was performed as previously described [escribed ,17,23,26Construction of Kaplan–Meier (KM) survival curves to determine prognostic effects. Publicly available microarray data  from 364 liver cancer patients  were analyzed using KMplotter [p-values, hazard ratios, and 95% confidence intervals were calculated by the software [Mplotter  to integMplotter . AdditioMplotter  to confisoftware .For IF analysis, one-way analysis of variance (ANOVA) or two-way ANOVA (for co-stained sections) was used to detect differences in the mean abundance of proteins among the various liver tissue types with Holm–Sidak’s multiple comparisons correction. Welch’s t-test was used to detect the differences in the mean values among two comparing groups. For bioinformatics analysis, Welch’s t-test was also used to detect the differences in the mean values among two comparing groups, while one-way ANOVA was used to detect the differences in the mean values among more than two groups. All IF graphs/plots/charts were constructed and produced using GraphPad Prism 9.3.1, while bioinformatics graphs/plots/charts were produced using either the Xena, KMplotter, or NCI-GDC built-in analysis software.Given our recent findings of the potential role of the CmPn signaling network in tumorigenesis of multiple types of cancers ,17,23,26We additionally performed socioeconomic status (SES) and diagnostic comparative profiling utilizing clinical data for patient samples without (PGRMC2 in CCAs), along with AFP, in our preliminary analysis , along with AFP . To evaluate differential expression of key CmPn players and their potential role as prognostic biomarkers, we also profiled AFP, an established liver cancer biomarker, to compare with our CmPn profiling analysis. For our first analysis, we utilized two databases in TCGA containing two categories of ‘normal’ tissues: “solid tissue normal” which are normal tissue sections taken adjacent to primary tumor tissue and “normal healthy tissue” taken from healthy individuals with no diagnosis of cancer. We observed significant differential expression patterns, in both HCCs and CCAs, for almost all CmPn players  including P53 signaling, viral carcinogenesis, colorectal cancer, base excision repair, and the Fanconi anemia pathway  in a pair-wise fashion, comparing normal healthy liver tissue to HCC primary tumor tissues, using publicly available RNAseq data. After analyzing DEGs between 110 normal healthy liver tissues and 369 HCC primary tumor tissues , we obsewith AFP . Our reswith AFP . Down-rewith AFP . Interes pathway . CCM1/2 (down in HCC), PAQR9 (up in HCC), PGRMC1/2 (up in HCC), nPRs (down in HCC), and AFP (up in HCC) in CCA primary tumors. Interestingly, CCM3 and PAQR5/7/8 were up regulated in CCAs  including PI3K-Akt, Notch, ECM–receptor interactions, regulation of actin cytoskeleton, tight junction, focal adhesion, and adherens junction signaling pathways  important in cancer signaling including Wnt, AMPK, PI3K-Akt, breast cancer, prostate cancer, ECM–receptor interactions, and proteoglycans in cancer signaling pathways , and cHCC-CCAs. After comparing all subtypes of CCA to HCC and cHCC-CCA clinical tumors, we observed significant differential expression of almost all CmPn members, as well as for CCM3 A. Recentfor CCM3 B, suggesAFP, among immune subtypes for both HCCs and CCAs. Clinical data available for HCC and CCA tumors included immune subtype classifications for wound healing (C1), IFN-gamma dominant (C2), inflammatory (C3), and lymphocyte depleted (C4) subtypes. Overall expression profiling of 355 HCC tumors, across all immune subtypes, revealed significant differential expression patterns for almost all CmPn players, along with AFP, excluding CCM2 and PAQR7/9 , characterized by elevated expression of angiogenic genes, a high proliferation rate, and a Th2 cell bias to the adaptive immune infiltrate, with IFN-γ dominant (C2) immune subtype, which has the highest M1/M2 macrophage polarization, a strong CD8 signal, high proliferation rate, and high T-cell receptor diversity . Analysind PAQR6 . Interesnd PAQR6  revealedInterestingly, when we repeated this analysis for CCAs, we did not observe any significant differential expression for key CmPn members , most liCCM3 and PAQR5/6, along with AFP (PGRMC1/2 (We next compared C1 subtype with the inflammatory immune subtype (C3), characterized by elevated Th1/Th17 genes, low to moderate tumor cell proliferation, lower levels of aneuploidy, and lower overall somatic copy number alterations than the other immune subtypes . Analysiwith AFP . We onlyPGRMC1/2 . InteresPGRMC1/2  revealedPAQR6  for PAQR6 , which iPAQR6  in CCAs PAQR5/6/8, along with AFP , characterized by a more prominent macrophage signature, with Th1 suppressed and a high M2 macrophage response . Analysiwith AFP . Signifiand CCM2 . Interesand CCM2  revealedPAQR6  for PAQR6 , which wPAQR6  in CCAs CCM3 and PAQR6/8, along with AFP  for with AFP . Signifiand CCM1 . Pathwayand CCM1  revealedPAQR5/6 and PGRMC1, along with significant up regulation of CCM2  for  of CCM2 . Pathway of CCM2  in CCAs PAQR5/8 along with AFP  for with AFP . Signifiand CCM1 . Pathwayand CCM1  revealedPAQR6 and PGRMC1  for d PGRMC1 . Pathwayd PGRMC1  in CCAs PAQR5/8, along with nPRs  for ith nPRs . Signifi  for CCM3 . PathwayCCM3  in CCAs AFP, in liver tumor tissues using patient clinical data including race, family cancer history, and vascular invasion, as well as follow-up data regarding new tumor events/sites and vital status. We first evaluated expression profiles based on the four major race categories available. Interestingly, for patients diagnosed with HCCs, significant differential expression of CCM1/2, PAQR7, PGRMC1/2, and nPRs, along with AFP, were observed across the four major races , intrahepatic recurrence (recurrence of cancer in the liver after hepatic resection), extrahepatic recurrence , new primary tumor, or no new tumor events. Interestingly, we observed significant differential expression in HCC patients for currence G. Next, patients H, while patients . Interespatients , but obspatients . Finallyl status I, but obl status . TogetheTo further examine the involvement of the CmPn network in liver tumorigenesis, we next examined differential expression patterns of key CmPn proteins across seven liver cancer subtypes and normal liver tissues . UtiliziFurthermore, we compared CCM3 and PAQR7 protein expression between HCC and cHCC-CCA , since cCCM1 and PAQR6 were the two members impacted with gain of function (GOF) alterations, while nPRs was the only member frequently impacted with a loss of function (LOF) alteration , we incorporated gene expression and clinical data simultaneously to generate Kaplan–Meier (KM) survival curves to understand the impact of the CmPn network on overall survival (OS) in liver cancer patients B. In eacRecent studies have highlighted the crucial role of the CmPn signaling network in the development of multiple types of cancer ,26,27,28Our findings revealed altered expression of several CmPn members (including CCM1/3 and PAQR7/8) between normal and tumor tissues and among different liver cancer subtypes, which was also observed for PGRMC1 D. AdditiWe utilized systems biology approaches to compare the alterations of key tumorigenesis pathways in CCAs and HCCs with normal healthy tissues, revealing similarities to those observed in our breast cancer omics analysis ,19,20,25Biomarkers play a crucial role in the prognosis and management of liver cancer, aiding in early detection and predicting patient response to therapy and the course of the disease ,61. WithThe current lack of reliable biomarkers for the diagnosis and prognosis of hepatic cancers underscores the need for identifying new biomarkers for various subtypes to improve early detection and treatment options for patients ,62,63,64CCM1 and nPRs were seen to be down regulated, at both the transcriptional and translational levels, in HCC and CCA tissues when compared to normal tissues, indicating that these CmPn members could be useful diagnostic biomarkers, but did not support its potential use as prognostic biomarkers in differentiating between cancer subtypes. Up-regulated levels of PAQR7 in HCC tissues only, at both the transcriptional  immune subtypes are characterized by elevated expression of angiogenic genes, while C2 (IFN-γ dominant) subtypes have high M1/M2 macrophage polarization . CirrhosWe next compared C1 immune subtype with the C3 (inflammatory) immune subtype, characterized by elevated Th1 and Th17 genes and low to moderate tumor cell proliferation . High leSimilarly, we compared C1 subtype to the C4 (lymphocyte depleted) subtype, which is classified as having suppressed Th1 levels and a high M2 response . An elevIn our analysis, we observed several shared up-regulated pathways in C2 subtype, for both HCCs and CCAs, including cytokine-mediated signaling, CAMs, and Th17 cell differentiation pathways . PreviouBased on our analysis, we observed shared up-regulated pathways in C2 subtype, for both HCCs and CCAs, including cytokine/chemokine receptor activity, CCR chemokine receptor binding, and hematopoietic cell lineage pathways . InteresFor our final comparison, we observed shared up-regulated pathways in C3 subtype, for both HCCs and CCAs, including integrin binding, general pathways in cancer, as well as AGE-RAGE signaling pathways . We alsoThese findings suggest the dysregulated pathways observed in both immune subtypes, as well as the differences in altered pathways between the subtypes, highlight the importance of understanding the immune landscape in hepatic cancers and the potential role of CmPn members as prognostic biomarkers.PAQR8 and PAQR9 are independent prognostic factors for HCC [PAQR9, which demonstrated increased expression resulted in decreased overall survival (As previously mentioned, differential expression of key CmPn members has been shown to be involved in the progression of multiple types of cancers, as well as leading to worse prognostic outcomes ,30,32,67 for HCC , which wThe findings in this study are significant because hepatic cancers are a major cause of cancer-related deaths worldwide, and early diagnosis and treatment can greatly improve patient outcomes. The identification of potential prognostic biomarkers and the discovery of disrupted signaling pathways and altered CmPn members in different subtypes of hepatic cancers can lead to the development of targeted therapies that could improve patient outcomes and potentially lead to better overall survival rates. Additionally, these results provide insights into the molecular mechanisms of liver cancer tumorigenesis, which may lead to a better understanding of the disease and new strategies for its prevention and treatment.


Alzheimer’s disease (AD) has no cure. Earlier, we showed that partial inhibition of mitochondrial complex I (MCI) with the small molecule CP2 induces an adaptive stress response, activating multiple neuroprotective mechanisms. Chronic treatment reduced inflammation, Aβ and pTau accumulation, improved synaptic and mitochondrial functions, and blocked neurodegeneration in symptomatic APP/PS1 mice, a translational model of AD. Here, using serial block-face scanning electron microscopy (SBFSEM) and three-dimensional (3D) EM reconstructions combined with Western blot analysis and next-generation RNA sequencing, we demonstrate that CP2 treatment also restores mitochondrial morphology and mitochondria-endoplasmic reticulum (ER) communication, reducing ER and unfolded protein response (UPR) stress in the APP/PS1 mouse brain. Using 3D EM volume reconstructions, we show that in the hippocampus of APP/PS1 mice, dendritic mitochondria primarily exist as mitochondria-on-a-string (MOAS). Compared to other morphological phenotypes, MOAS have extensive interaction with the ER membranes, forming multiple mitochondria-ER contact sites (MERCS) known to facilitate abnormal lipid and calcium homeostasis, accumulation of Aβ and pTau, abnormal mitochondrial dynamics, and apoptosis. CP2 treatment reduced MOAS formation, consistent with improved energy homeostasis in the brain, with concomitant reductions in MERCS, ER/UPR stress, and improved lipid homeostasis. These data provide novel information on the MOAS-ER interaction in AD and additional support for the further development of partial MCI inhibitors as a disease-modifying strategy for AD. Alzheimer’s disease (AD) is a devastating neurodegenerative disorder without a cure. The accumulation of misfolded amyloid beta (Aβ) peptides and hyperphosphorylated microtubule-associated tau protein (pTau) represents two hallmarks of AD. However, an incomplete understanding of the underlying molecular mechanisms hampers the development of disease-modifying strategies. The limited success of clinical trials focused on Aβ and pTau-reducing therapies emphasizes the need to identify new targets and therapeutic approaches. AD is a multifactorial disease that has developed over time, where neuroinflammation, altered energetics, and metabolism, together with genetic and environmental contributions, play important roles . Abnorma2+) signaling, govern biosynthesis of macromolecules , improve innate immunity, and induce apoptosis  dissolved in drinking water ad lib) or vehicle-containing water (0.1% PEG) starting at 9 months of age , which reflects the “length to width ratio”. The sphericity represents the adherence of the object to that of a sphere and was calculated using the equation: Quantitative analysis of mitochondrial morphology was conducted using the label analysis and measure tool functions in the Amira project view to obtain parameters related to mitochondrial shape  and cytoplasmic (CY supernatant) fractions. The MT pellet was washed twice with ice-cold MIBA buffer before it was resuspended in the lysis buffer. Three mice from each experimental group were taken for analysis.This protocol was adapted from  with a fn = 2–3 mice per group) using Western blot analysis. Samples were homogenized and lysed using 1× RIPA buffer plus inhibitors. Total protein lysates (20 µg) were separated in equal volumes on 4–20% Mini-PROTEAN TGX™ Precast Protein Gels  and transferred to a PVDF membrane (cat. # 1620177). The following primary antibodies were used: Drp1 , phospho-Drp1 S616 , OPA1 , Mfn1 , Mfn2 , Vdac1 , Tfam , Tfeb , LC3b , Pink1 , Parkin (PRK8) , and β-Actin . The following secondary antibodies were used: Peroxidase (HRP) Anti-Rabbit IgG (H+L) Goat Secondary Antibody ; and Peroxidase AffiniPure Goat Anti-Mouse IgG (H+L) . Band quantification was performed using the ChemiDoc Imaging System from Bio-Rad. Data analysis was performed using Image J software.Levels of proteins were determined in cortico-hippocampal regions of the brain or enriched mitochondria fractions from cortico-hippocampal brain regions obtained as described above from vehicle and CP2-treated NTG and APP/PS1 mice (n = 5 per group) has been published here . N. N34]. NCompared to traditional 2D transmission EM TEM, c, 3D EM For 3D EM reconstruction, 20–40 serial sections were generated using SBFSEM, stacked, aligned, and visualized using Amira b or RecoData were collected from NTG and APP/PS1 mice treated with vehicle or CP2 to determine the effect of treatment on mitochondrial shape, length, and the interaction with the ER . The repWe have previously shown that AD patients and multiple mouse models of AD, including APP/PS1 mice, have increased MOAS formation in the brain . MOAS arWe found that in NTG mice, dendritic mitochondria in the hippocampus were predominantly of Type II (~70%), with a lower percent of Types I (~5%) and III (and ~15%) c. NTG miExtensive MERCS formation in AD was shown to facilitate Aβ production, abnormal lipid homeostasis, ER stress, and apoptosis ,58,59. WThe significant reduction in MOAS after CP2 treatment in APP/PS1 mice could be associated with improved glucose uptake and utilization in the brain, detected using in vivo 18F-fluorodeoxyglucose-positron emission tomography (FDG-PET) ,34. HoweWe further assessed the effect of CP2 treatment on the expression of mitochondrial fission and fusion proteins essential for mitochondrial turnover. Our earlier studies revealed that the most effective way to determine changes in these proteins was to assay mitochondrial fractions isolated from the brain regions of interest ,28. IndeNupr1) involved in the regulation of autophagy-induced apoptosis through FOXO3 interaction, UPR, and the integrated stress response [Clu). The gene product of Clu functions as an extracellular chaperone that prevents aggregation of proteins [Alox5), Fc gamma receptor IIIa (Fcgr3), Fc gamma receptor IIb (Fcgr2b), and wolframin ER transmembrane glycoprotein (Wfs1), which are involved in mediating the inflammatory response, dendritic cell migration, apoptosis, and intracellular calcium signaling [To further investigate the mechanism of CP2 action, we utilized the RNA-seq data previously generated using cortico-hippocampal brain tissue from vehicle- and CP2-treated NTG and APP/PS1 mice from the same cohorts that we used in the current study . We idenresponse ,61. Nuprresponse ,63. Anotproteins  and inhiproteins . Moreoveproteins . Interesproteins . An intrproteins . Other gignaling ,70,71,72ignaling . Nfe2l2, Casp8, Il1b, Tnfrsf23, Pmaip1, and Txnip was increased in APP/PS1 mice and reduced after CP2 treatment, while the expression of Hspa5 was reduced in APP/PS1 mice and increased after CP2 treatment (Nfe2l2), caspase 8 (Casp8), interleukin 1 beta (Il1b), tumor necrosis factor receptor superfamily, member 23 (Tnfrsf23), phorbol-12-myristate-13-acetate-induced protein 1 (Pmaip1), and thioredoxin interacting protein (Txnip) , mitochondrial dynamic protein MFN1 (Mfn1), and mitophagy-related protein Pink1 (Pink1), consistent with the Western blot analysis  f. Importat MERCS ,77,78,79analysis . Collect2+ homeostasis, and ER stress, among others. Since no biomarkers are available to clearly define at what stage of the disease what mechanism becomes affected, a successful approach to treatment may require targeting multiple pathways at the same time. Such a polypharmacy approach is very difficult to implement given the complicated etiology of AD. However, non-pharmacological approaches, such as exercise, have been shown to improve cognitive function, reduce inflammation and oxidative stress, and positively affect other major mechanisms underlying AD [AD is a complex disorder, with multiple pathways affected during the disease’s development. These include inflammation, dysfunction of mitochondrial dynamics, function, and bioenergetics, oxidative stress, altered protein and Calying AD . We idenlying AD ,36,37,44lying AD ,36 or folying AD . We conslying AD ,81,82. Mlying AD ,37. Amonlying AD ,83,84. TOne of the novel observations generated in this study was the demonstration that MOAS are increasingly associated with the ER, forming extensive MERCS. Until now, this phenomenon has not been described in the context of AD. Our data suggest that MOAS, a mitochondrial phenotype formed in response to energetic stress, has extensive interactions with the ER, greater than any other type of mitochondria. Currently, the precise mechanism of MOAS formation is not well understood, and it is unclear why MOAS are increasingly associated with the ER membranes. However, given the important role the ER plays in mitochondrial dynamics, fission in particular, such interaction may be instrumental for MOAS formation, promoting fission arrest under conditions of energetic stress, as well as MOAS elimination after stress relief . Our pre31P Nuclear Magnetic Resonance (31P NMR) Spectroscopy and in primary mouse neurons in vitro [Partial restoration of brain energy homeostasis after CP2 treatment reduced MOAS formation and their interaction with the ER. CP2 treatment also reduced the number of small mitochondria, whose formation is commonly associated with mitochondrial dysfunction in AD . Most ofin vitro . These dOur findings further demonstrate that CP2 treatment may not only be beneficial for AD but also improve health span, delaying the onset of AD in aging individuals. We demonstrated that the mitochondrial MERCS coverage was reduced by CP2 treatment in APP/PS1 mice to a level below that observed in age-matched 24-month-old NTG mice. Since increased MERCS were found to be associated with senescence and aging , this ma2+ signaling, which also might have contributed to the increased neuronal survival and cognitive protection [Another important outcome of this study is the validation of the translational value of APP/PS1 mice that recapitulate changes in mitochondrial morphology and MERCS hypergeneration found in AD patients ,92. Manyotection . Transcrotection . Here, wotection . Recent otection . Our datotection  further otection . Restored MERCS and mitochondrial morphology, improved lipid and glucose homeostasis, and reduced UPR and ER stress in APP/PS1 mice after CP2 treatment coincide with reductions of Aβ and pTau ,28. The


Primary HPT (PHPT) is a common disorder, affecting approximately 1% of the general population. Parathyroid adenomas emerge as non-familial sporadic in 90% of cases. The aim of this review is to give a detailed update of molecular genetics of sporadic parathyroid adenoma reported in international literature.A bibliographic research was conducted in PubMed, Google Scholar, and Scopus.Seventy-eight articles were included in our review. CaSR, MEN1, CCND1/PRAD, CDKI, angiogenic factors like VEGF, FGF, TGFβ, and IGF1, and apoptotic factors are important genes in parathyroid adenomas pathogenesis that have been established by several studies. A huge list of proteins is differently expressed in parathyroid adenomas measured by Western Blotting, MALDI/TOF, MS spectrometry, and immunohistochemistry. These proteins take part in several cell processes such as cell metabolism, cytoskeleton structural stability, cell oxidative stress regulation, cell death, transcription, translation, cell connection, and cell signaling transmission, while they can be found over- or underexpressed in abnormal tissues.This review gives a detailed analysis of all reported data on genomics and proteomics of parathyroid adenoma. Further studies should be applied on understanding parathyroid adenoma pathogenesis and introducing new biomarkers for early detection of primary hyperparathyroidism. Primary HPT (PHPT) is a common disorder, affecting approximately 1% of the general population. It is more frequent in women, with a women-to-men ratio of 3–4:1. PHPT results from a single parathyroid adenoma in 80%–85% of cases, multi-gland hyperplasia in 15%, and cancer in 1%. In the majority of cases, PHPT remains asymptomatic and is diagnosed incidentally during routine blood examination. In cases of symptomatic disease, musculoskeletal, urinary, gastrointestinal, neural, and cardiovascular systems are affected. Surgical treatment is the only curative treatment and is indicated in cases of symptomatic disease and in asymptomatic if at least one of these conditions is fulfilled: (a) age <50 years old; (b) serum Ca >1 mg/dl (>0.25 mmol/L); (c) T-score below -2.5 in one of the following bone regions: lumbar spine, total hip, femoral neck, or 1/3 distal radius bone or detection of asymptomatic vertebral fracture by any imaging method; (d) creatinine clearance <60 cc/min; (e) 24-h urine calcium >300 mg/d (<10 mmol/d) together with increased risk of kidney stones based on biochemical stone risk analysis, presence of asymptomatic nephrolithiasis, or nephrocalcinosis detected by imaging methods . At last, 78 articles were included in our systematic review.The first genetic studies on the pathogenesis of sporadic parathyroid adenoma were conducted in 1990, and until now a large amount of genes are associated with adenoma development. These genes are mainly regulators of cell cycle, growth factors, apoptosis factors, death receptors, and signal transduction molecules. A great variety of genetic mutations, especially somatic ones, have been observed such as base insertions, deletions, and substitutions , 7. Tab3.1Alteration in CaSR  signaling pathway, regulating parathormone (PTH) secretion, has been identified. CaSR protein is a plasma membrane G-protein and acts as a receptor on chief parathyroid cell surface. It binds serum calcium and activates the signaling pathway, which includes the phospholipase Cβ and the protein kinase C, resulting in suppression of PTH secretion. In addition, vitamin D receptors are proliferated, binding 1.25 dihydroxivitamin D and also suppressing PTH secretion. CaSR gene, located in chromosome 33q13.3–21, spans about 103 kb and has eight exons. Exons 2–7 encode a CASR protein of 1,078 amino acids , 10. CaSMEN1 gene, which plays an important role in hereditary disease, is an oncosuppressor gene, located in chromosome 11q13 and aims menin protein through Wnt/β-catenin signaling pathway , 10, 13.CCND1/PRAD1 gene is a proto-oncogene, located in 11q13 chromosome, nearby another gene that regulates PTH secretion, and is translated to cyclin D1 protein. Cyclin D1 is a regulator factor of G1-S cell cycle phase and binds to CDK4 and CDK6 which belongs to the same family . Its invMutations in CDKI genes (cyclin D1 inhibitors) are involved in CCND1 gene regulation. CDKN1B, CDKN1A, CDKN2B, and CDKN2C germline mutations have been recorded . GermlinDuan et al. found mutations related to parathyroid adenoma development in 5% of ZFX gene aiming cyclin D1, in 2%–5% of CTNNB1 gene, encoding β-catenin, in 1% of EZH2 gene, accelerating β-catenin and <1% of POT1 which is a genome stabilizer .EZH2 gene encodes histone methylase H3K27 and a rare mutation (p. Y641N) has been correlated to parathyroid adenoma development . Gene onZFX gene encodes a transcription factor related to cell renewal maintenance. Somatic mutations have been identified in 4.6% in abnormal parathyroid tissue, while gene oncogenesis mechanism remains unclear .Wnt/β-catenin/CTNNB1 signaling pathway is enrolled in tumorigenesis, but its role in parathyroid adenoma has not been yet elucidated. β-catenin is the first molecule in Wnt pathway. The Costa-Guda et al. study in 97 patients with sporadic parathyroid adenoma for mutations in exon 3 on CTNNB1 gene was negative for novel mutations except for S37A mutation which is already known from previous studies . S37A muErb/ηer-1/EGFR gene is associated with other endocrine disorders or tumors such as breast cancer. DNA from 33 patients with PHPT was assessed using real-time PCR concluding in one amplification of ErbB-1/Her-1/EGFR gene, two deletions of ErbB-2/Her-2 gene, and six deletions of ErbB-4/Her-4 gene, which was reasonably selected as the most appropriate for further investigation .SMAD3 gene is located in chromosome 15 and encodes a TGF-β pathway molecule that binds to menin. Due to this association with menin, SMAD3 mutations leading to abnormal development of parathyroid chief cells were carefully investigated. Indeed, loss of heterozygosity was detected in 24% of parathyroid adenomas, but SMAD3 cannot be characterized as oncosuppressor gene as clonal mutations were not found , and in 4q21-28 and 8p22-23 (in 4/14 patients). Deletions were found in chromosomes 11 and 20 (20q12-13) in 2/14 cases , and one somatic mutation in the EZH2 gene were detected in a Chinese population study. In this study, whole genome sequencing was applied in 22 blood samples from patients with sporadic parathyroid adenoma .A germline mutation (deletion) in CDC73/HRPT2 gene was verified in 35% of patients with sporadic parathyroid adenoma by real-time PCR. This gene is involved in Hyperparathyroidism-jaw tumor, so it is recommended as a possible oncogene in sporadic parathyroid adenoma cases . CDC73/HBy comparative genomic hybridization (CGH) application, genome from 53 parathyroid adenomas was analyzed, and both insertions in 16p and 19p genes and deletions in 11p and 11q genes were detected. These findings introduce new oncogenes and oncosuppressor genes involved in parathyroid adenoma pathogenesis .By PCR, macroarray, protein, and mRNA expression techniques, additional genes in parathyroid adenoma tissues were inserted in an involved gene list: APOLLON, BCL2, CK19, CK18, CHROMOGRANIN-A, BAX, PARATHORMONE, ATM,MDM2, CK8, CYCLIN D1, SYNAPTOPHYSIN, FLIP και TRAIL . EpigeneIn a review by Westin et al. in 2016, β-catenin is an essential part of molecular pathways in parathyroid adenoma pathogenesis. MEN1, HRPT2, EZH2, RASSF1A, HIC1, WT1, APC, SFRP1, SFRP2, SFRP4, SFRP5, and LRP5 genes are related to β-catenin by promoting or inhibiting its production. Wnt/β-catenin genetic pathway is suggested as the main pathway in sporadic parathyroid adenoma development, although further studies is required to verify its role .Further molecular pathways involved in parathyroid adenoma pathogenesis include angiogenic factors. VEGF, FGF, TGFβ, and IGF1 factors were found to be correlated with parathyroid adenoma. VEGF has a pre-angiogenic action and takes part in cell proliferation. FGF factor is increased in adenomas, as it regulates cell cycle and cell development. TGFβ is also increased in adenomas, related to several cell processes. IGF1 is involved in cell proliferation and stands for the main mediatory factor in PTH anabolic role on bones .Apoptosis factors also seem to be associated with parathyroid adenomas. These molecular pathways include anti-apoptotic and apoptotic agents, of which action is altered in adenoma development. TRAIL protein is bound to DR4 and DR5 death receptors and has an anti-cancer role; although TRAIL protein can create complexes with caspase-8 and FADD, leading to opposite action. TRAIL protein is highly expressed in parathyroid adenomas. FAS protein is a death receptor participating in DISC  formation and its production is elevated in adenomas. BCL2 gene promotes caspase activation and mitochondrial external membrane permeability and was found decreased in adenomas. MDM2, negative regulator of p53, is elevated in adenomas and acts as oncogene .Lu et al. in 2018 studied genetic factors involved in parathyroid adenoma pathogenesis applying immunohistochemistry, real-time PCR, and spectrometry in both histological types of parathyroid adenomas (oxyphil and chief cell adenomas). CaSR, VDR, PTH, MEN1, FGFR1, CNND1, CDKN1B, GCM2, CYP27B1, CYP24A1, PTHLH, and NDUFA13 expression has no statistical significant difference between two groups. VDR and PTH gene expression in parathyroid adenomas ranges from very low to high compared to normal tissue where PTH and VDR are highly expressed .Although several genes have been detected in sporadic parathyroid adenoma, genetic testing in sporadic disease is not recommended, as the majority of mutations is somatic and is developed after disease progression . On the 3.2In international literature, studies of proteomic analysis of parathyroid adenoma tissues have been reported. Proteins differently expressed in the abnormal tissues have been measured by Western Blotting, MALDI/TOF, MS spectrometry, and immunohistochemistry. There are a vast number of proteins which expression is modified in parathyroid adenomas. Except for the quantitative measurement of these proteins, studies also focus on their functional role and their localization in cells, in order that genetic pathways related to these proteins are detected. The functional role of proteins usually is translated as parts of cell metabolism, cytoskeleton structural stability, cell oxidative stress regulation, cell death, transcription, translation, cell connection, and cell signaling transmission , 36–39. In sporadic parathyroid adenomas, localization of several proteins is in the cytoplasm, as Akpinar et al. reported . DiffereANXA-2, ATP5H, and LMNA are examples of proteins detected in parathyroid adenomas, fact that has been verified by three different studies , 39, 40.VDAC1, ANXA5, ANXA2, and S100-A11 proteins are increased in parathyroid adenomas. VDAC1 is involved in the mitochondrial molecule release process that intensifies apoptosis, and its overexpression in adenomas is found to cause PTH release to peripheral circulation. Annexin role in oncogenesis is under intense investigation. ANXA-5 is found to be related to apoptotic processes, while ANXA-2 enhances tumor metastasis by reinforcing cell detachment and translocation.COX-5A, PARK-7, PRDX-3, HSP90, HSP60, and CCT5 proteins are also found overexpressed in parathyroid adenomas. These proteins are involved in early cell response to oxidative stress, fact that characterizes adenomas which have intense cell proliferation and neoangiogenesis. BSPRY protein, related to calcium receptors, is underexpressed in parathyroid adenomas, leading to decreased inhibitory role on calcium receptors. Its role remains unclear . ProlactDonaldio et al., in their proteomic analysis, used paraffin embed parathyroid adenoma tissues which were reconstituted with formalin solution. Ten from 20 proteins detected in their study were found in these tissues, which is of great importance for future studies on specially processed adenoma samples in order that new biomarkers are discovered .4This review gives a detailed analysis of all reported data on genomics and proteomics of parathyroid adenoma. Different signaling pathways are involved in adenoma development. Wnt/β-catenin, MAPK, Ras, IP, TGF-β, and PLC have already been established, including many genes and proteins with different functional roles. Further studies should be applied on understanding parathyroid adenoma pathogenesis and introducing new biomarkers for early histological biomarkers to predict recurrence in other parathyroid glands.ACho, TP, AChe, ACha: Conceptualization. ACha, CA, KB: Data curation. ACho, AChe, ACha: Formal analysis. SM, TZ, ACho, CA, KB, KD: Investigation. ACho: Methodology. TP, AChe, ACha: Supervision. TP: Validation. ACho: Writing - original draft. TP, AChe, ACha: Writing - review and editing. All authors contributed to the article and approved the submitted version.


Neuroblastoma (NB) is the most common extracranial solid pediatric cancer, with highly observed metastasis at diagnosis. Primary NBs generally have favorable outcomes with current therapies; however, high-risk, metastatic NBs are resistant and result in relapsed disease. Patients with metastatic NBs, thus, have a lower overall survival. Thus, to develop effective therapies for metastatic NBs, it is essential to better understand the biology of metastasis in NB. This requires the utilization of pre-clinical animal models, importantly, immunocompetent models that have an intact immune system and better reflect the disease physiology. However, currently, there are few good animal models available for studying HR-NB metastasis. This study aimed to generate efficient and appropriate mouse models to study HR-NB metastasis. Our newly developed and validated mouse cell lines are relevant and promising tools to study (high-risk) HR-NB metastasis and identify novel therapeutic targets for metastatic NBs; thus, they could benefit the NB research community.NB, being a highly metastatic cancer, is one of the leading causes of cancer-related deaths in children. Increased disease recurrence and clinical resistance in patients with metastatic high-risk NBs (HR-NBs) result in poor outcomes and lower overall survival. However, the paucity of appropriate in vivo models for HR-NB metastasis has limited investigations into the underlying biology of HR-NB metastasis. This study was designed to address this limitation and develop suitable immunocompetent models for HR-NB metastasis. Here, we developed several highly metastatic immunocompetent murine HR-NB cell lines. Our newly developed cell lines show 100% efficiency in modeling experimental metastasis in C57BL6 mice and feature metastasis to the sites frequently observed in humans with HR-NB (liver and bone). In vivo validation demonstrated their specifically gained metastatic phenotype. The in vitro characterization of the cell lines showed increased cell invasion, acquired anchorage-independent growth ability, and resistance to MHC-I induction upon IFN-γ treatment. Furthermore, RNA-seq analysis of the newly developed cells identified a differentially regulated gene signature and an enrichment of processes consistent with their acquired metastatic phenotype, including extracellular matrix remodeling, angiogenesis, cell migration, and chemotaxis. The presented newly developed cell lines are, thus, suitable and promising tools for HR-NB metastasis and microenvironment studies in an immunocompetent system. Neuroblastoma (NB) is the most common extracranial solid cancer in children, and it accounts for around 15% of cancer-related deaths in pediatric patients ,3,4. AppMetastasis is a multistage event that is largely regulated via the microenvironment, consisting importantly of immune cells at the primary tumor location, as well as secondary sites. The utilization of immunocompetent pre-clinical models is, thus, essential to examine the progression of metastasis. Current pre-clinical HR-NB models include the xenograft implantation of human NB cells in immunocompromised mice, injecting immunocompetent murine cell lines  in their respective syngeneic mouse models, and transgenic mice that develop spontaneous tumors ,11,12,13The 9464D NB cell line was derived from the spontaneous tumor of the TH-MYCN mouse and efficiently forms primary tumors in subcutaneous or orthotopic models . This 94We developed several cell lines from 9464D-derived metastatic tumors (C57Bl/6 background) that model experimental metastasis in immunocompetent mice with 100% efficiency. Here, we present two highly metastatic cell lines (M1 and M2) derived from the liver metastasis of 9464D cells and several M1 variants derived from different metastatic tumors in different locations. Given sufficient time, these cell line models developed metastasis in all the IV-implanted animals. This study characterizes M1 and other newly developed 9464D variant cell lines, comparing them to the original 9464D. In vivo validation showed that the newly developed cell lines not only have a significantly higher metastatic potential but also exhibit organ-specific metastasis, particularly in the bone and liver, two prominently observed sites of metastases in HR-NB patients . In vitrv/v fetal bovine serum  and 100 units/mL of penicillin and streptomycin . Stable 9464D-luciferase reporter cells were generated as previously mentioned, using pCDH-EF1-Luc-p2a-tdTomato (RRID: Addgene_72486) lentiviral transduction, followed by tdTomato fluorophore sorting. The cells were then verified for luciferase activity using the Promega luciferase assay system .Mouse neuroblastoma cell line 9464D was cultured in Dulbecco’s modified Eagle’s medium , supplemented with 10% Initially, in order to determine a workable cell number for experimental metastasis for our previous study, 9464D (Luc-tdT) cells were IV-injected at different cell titers  in C57Bl/6J mice   was IV-injected into C57Bl/6J mice. These mice were monitored for the growth of experimental metastasis via in vivo bioluminescence imaging. Tumors from different locations, relevant to the prominently observed human primary and metastatic HR-NB sites, were isolated and processed as mentioned above. The obtained homogeneous HR-NB cell lines were validated using MYCN expression and named M1-1 (derived from lymph nodes), M1-2 (bone and the hindlimb), M1-3 (the stomach/intestine), M1-4 (the thoracic region (ganglia)), M1-5 , M1-6 , and M1-7 (the lumbar vertebrae/ganglia). All the lines were cultured using the same DMEM medium as above. All the above parental and generated cell lines were regularly tested for mycoplasma contamination  and were cultured for less than a month for the experiments.n ≥ 4 animals/group) for all experiments. No blinding was performed. For primary tumor growth characterization, 0.5 × 106 9464D, M1, M2, or M1-variant cells were injected subcutaneously, and the mice were monitored for tumor growth and survival. Tumor sizes  were measured twice a week until they reached the experimental endpoint size of ≥30 mm.Six-to-eight-week-old C57Bl/6J mice were used, and the animals were randomly assigned to each group , followed by monitoring these mice for metastasis growth using an in vivo bioluminescence imaging system. The mice were anesthetized using 2.5% isoflurane  and injected with 0.15 mg/g body weight of D-Luciferin  dissolved in PBS 10 min prior to imaging. The mice were imaged once or twice a week. The animals were followed for survival analysis. Humane euthanasia/death was the experimental endpoint for these metastasis experiments. All in vivo mouse studies were approved by the Institutional Animal Care and Use Committee (IACUC) at the Pennsylvania State University College of Medicine.To validate their efficiency for experimental metastasis, 1 × 10The 9464D, M1, M2, and M1 variants’ M1-2, M1-5, and M1-6 cells were seeded at 5000 cells/well in 96 well plates for proliferation assays. Live-cell phase contrast images were obtained using a 10× objective lens (three images per well), and cell confluence was analyzed using IncuCyte Live Cell Analysis (v2019B) software. For migration assays, 40,000 cells/well (100 μL/well) were seeded in IncuCyte ImageLock 96-well plates , and after 12–14 h, scratches were made using the IncuCyte 96-well Wound Maker Tool (Sartorius 4563), as per the manufacturer’s protocol. Images were obtained every 2 h for 72 h, and relative wound density was quantified using the Incucyte Cell Migration Analysis software (Sartorius 9600-0012).v/v) formaldehyde (in PBS), followed by 100% methanol. The inserts were then stained with 0.25% crystal violet (in 20% methanol), rinsed, dried, and analyzed for invaded cell numbers under a 20× phase contrast microscope. Images from 7–8 random areas were acquired using the Biorad ZOE Fluorescent cell Imager. The area of invaded and migrated cells from the Matrigel and control inserts, respectively, was quantified using the ImageJ software (https://imagej.nih.gov/ij/index.html (accessed on 22 January 2021)). Cell invasion data is expressed as % invasion through Matrigel matrix inserts relative to migration through the control inserts (formulae: % invasion = area of invaded cells through Matrigel inserts/area of migrated cells through control inserts × 100).Invasion assays were performed using the Corning Biocoat Matrigel invasion chambers (Corning 354480) and control inserts (Corning 354578), as per the manufacturer’s protocol. Briefly, 25,000 cells in 500 μL of serum-free media were seeded in the inserts, and complete media (+10% FBS) was added to the wells of the companion plate as a chemoattractant. After 48 h, the inserts were fixed and permeabilized by subjecting them to 4% (https://imagej.nih.gov/ij/index.html (accessed on 22 January 2021)).Single cells were seeded at a density of 1000 cells/well in 6 well plates and allowed to grow until the appearance of prominent colonies. On day 6, the wells were washed twice with PBS, and the colonies were fixed using 4% formaldehyde, followed by 100% methanol for 5 min each. The colonies were then stained with 0.25% crystal violet (in 20% methanol) for 20 min at room temperature (RT) and rinsed with tap water. The plates were dried on a bench top for 2 days, and images were acquired using the ChemiDoc MP imaging system  and analyzed using ImageJ  bovine serum albumin (BSA) in tris buffered saline (TBS) (+0.1% Tween-20). The primary antibodies included N-Myc , β-Actin , and horseradish peroxidase-conjugated secondary antibody: anti-rabbit IgG .Western blotting was performed as previously described . Briefly5 cells were plated in 6 well plates in 2 mL of DMEM media (+10% heat-inactivated FBS (Gibco), 2 mmol/L of L-Glutamine (Corning), 100 U/mL of penicillin, and 100 μg/mL of streptomycin (Corning)). After 24 h, the cells were treated with 1000 U/mL of IFN-γ . Forty-eight h post-treatment, the cells with or without IFN-γ were harvested, washed with a FACs buffer (PBS + 2% FBS), and centrifuged at 300× g. The pelleted cells were resuspended in around 50 µL of FACs buffer and stained with APC anti-mouse H-2Kb/H-2Db  antibody for 30 min at RT in the dark. Following a washing step (300× g for 5 min), a drop of DAPI  was added to the cell suspensions, and data were acquired using a ThermoFisher Attune NxT flow cytometer. The data were analyzed using FlowJo v.10.8.1.For MHC-I and PD-L1 expression analysis, 2.5 × 106 cells were resuspended in 200 µL of PBS and washed once via centrifuging . The pelleted cells were resuspended in 150 µL of FACs buffer (PBS with 2% FBS + 0.1% sodium azide), and centrifuged as above. The cell pellets were resuspended in 50 µL of FACs buffer and stained with FITC anti-human Ganglioside GD2  for 30 min on ice in the dark. The cells were then washed  twice with 150 µL of FACs buffer, fixed with 200 µL of 2% paraformaldehyde for 20 min on ice, and, at the final step, resuspended in 300 µL of FACs buffer. As a positive control, 9464D-GD2 cells, generated via GD2/GD3 synthase  lentivi® for Illumina RNA UD Indexes Set C, Ligation, Illumina) were incorporated into the adaptors for multiplexed high-throughput sequencing. The final product was assessed for its size distribution and concentration using a BioAnalyzer High Sensitivity DNA Kit . The libraries were pooled and sequenced via Illumina NovaSeq 6000 (Illumina) to get, on average, 25 million paired-end 50 bp reads, according to the manufacturer’s instructions.Three biological replicates of 9464D and M1 cells were collected, and total RNA isolation was done using a TRIzol reagent , as per the manufacturer’s protocol, before being subjected to RNA-seq. RNA-seq libraries were prepared in the Penn State College of Medicine Genome Sciences core (RRID: SCR_021123) using the Illumina Stranded mRNA Prep Ligation kit , as per the manufacturer’s instructions. Briefly, polyA RNA was purified from 200 ng of total RNA using oligo (dT) beads. The extracted mRNA fraction was subjected to fragmentation, reverse transcription, end repair, 3′– end adenylation, and adaptor ligation, followed by PCR amplification and magnetic bead purification . The unique dual index sequences (IDTMus musculus GRCm39 reference genome using STAR v.2.7.3a with the quantMode Gene Counts option to generate read counts per gene [t-tests relative to a threshold (TREAT) method described by McCarthy and Smyth with a significant absolute log2 fold change = 1 [http://metascape.org (accessed on 17 February 2023)) [De-multiplexed and adapter-trimmed sequencing reads were generated using Illumina bcl2fastq (released version 2.18.0.12), allowing no mismatches in the index read. The reads were aligned with the per gene . Differeper gene . Brieflyange = 1 . Differeange = 1  R packagange = 1 ,27, and ange = 1 . Signifiy 2023))  to identGraphPad Prism 9 was used for statistical analysis. One-way ANOVA was used to compare multiple groups, and a non-linear regression model was used to compare tumor growth rates. The data are presented as means ± SEMs. Survival curves were generated using Kaplan–Meier survival analysis. Statistical significance is indicated on the graphs, as well as in the figure legends (significance level: α = 0.05).+ 9464D cells injected by IV. In addition, the M1 and M2 cells exhibited the overexpression of N-Myc, similar to 9464D  injected with 9464D cells, only two developed metastatic tumors. Liver tumors from both these mice were isolated and processed to generate established lines—named M1 and M2, respectively A. Their to 9464D , validatInitially, the newly generated cell lines were examined for their ability to form primary tumors, comparing them to 9464D cells. For M1, M2, and all M1-derived subcutaneous models except M1-4, tumor growth was observed in all mice inoculated subcutaneously. Specifically, M1-2 and M1-6 exhibited significantly faster tumor growth over the period of monitoring, with the appearance of palpable tumors at week 1 post-cell-injections A. IrrespBased on their derived locations  and relevance to the prominently observed metastatic sites in HR-NB patients, we further chose M1-2, M1-5, M1-6, and M2, along with M1, to examine their potential to generate metastases after IV injections. As previously demonstrated , the M1 The 9464D cell line is derived from the spontaneous tumor of the TH-MYCN mice and, thus, expresses high levels of MYCN [In addition, we evaluated the cellular phenotypes of these cells in culture, comparing them to 9464D cells. Cell proliferation, migration, invasion, colony formation, and anchorage-independent growth are the standard functional assays to assess the tumorigenic behavior of cancer cells. M1, M2, and M1-derived cells were examined for these in vitro cellular phenotypes. Both M1 and M2 cells showed reduced cell proliferation and migration compared to 9464D. Similarly, all the M1 derivatives exhibited a lower in vitro proliferation and migration rate relative to 9464D cells . While MHigh GD2 expression is a characteristic of human NBs and is a potent target for current clinical immunotherapies for NB. Nearly all human HR-NB cell lines have high GD2 expression; however, most of the murine HR-NB cell lines, including 9464D, express very low levels of GD2. Furthermore, previous studies have shown the dynamic nature of GD2 expression on 9464D cells . To obtaTo determine the potential mechanisms responsible for the acquired metastatic ability, we compared transcriptomes of M1 and parental 9464D cells utilizing RNA-seq analysis. We found a substantial cluster of significantly up- and downregulated genes in M1, relative to the 9464D cells. A stringent FDR of 0.05 (5%) was set to identify the significant hits. These top-most differentially regulated (686) genes consisted of 308 upregulated and 378 downregulated genes A. FurtheIn contrast, the downregulated genes were linked with biological processes, such as the positive regulation of apoptosis, the negative regulation of cell migration, negative chemotaxis, and the negative regulation of blood vessel endothelial cell migration and angiogenesis . OverallIn this report, we present novel, highly metastatic HR-NB syngeneic mouse models. These clinically relevant murine HR-NB models feature metastasis to the organs that are characteristic of human HR-NB metastasis. Due to the lack of a spontaneous metastasis model in NB, an experimental metastasis approach is the next best available strategy for studying metastasis in NB. The TH-MYCN transgenic mouse model and the respective derived HR-NB cell line 9464D closely resemble human NBs in terms of gene expression profiles and tumor phenotype . HoweverThe in vitro cellular characteristics, such as proliferation, migration, or colony formation, of these cell lines were not higher than those of the 9464D cells. But the specifically acquired anchorage-independent growth phenotype of the newly developed cells may indicate their gained ability to survive the fluid shear flow and proliferate in circulation, eventually colonizing and forming metastases at secondary sites. The anchorage-independent growth ability is reflective of surviving detachment, the initial step of metastasis in vivo ,32. CancOne of the major biological processes upregulated in M1 cells is the increased production of Interlukin-1α (IL-1α). Previous studies have shown the tumor-promoting function of inflammatory cytokine IL-1α in multiple cancer models, such as for liver, lung, ovarian, pancreatic, and head and neck cancers ,36,37,38The significantly higher invasive ability of M1 cells, along with the observed associated gene expression profile, further explains the aggressive nature of M1 in vivo. Gene ontology analysis identified extracellular matrix (ECM) and extracellular structure organization, tissue remodeling, actin cytoskeleton organization, and cell adhesion processes among the top regulated biological processes in M1 cells. ECM degradation and remodeling is one of the vital events in cancer metastasis, essential for cancer invasion. Importantly, MMP15, MMP28, ADAMTS12, ADAM19, COL4A1, and COL24A1 are some of the upregulated genes associated with these processes.Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases that play multidimensional roles at different stages of cancer progression, specifically by remodeling the existing ECM to a tumor-specific ECM. MMPs have been known to promote metastasis by regulating the proteolytic degradation of ECM components, promoting invasion and angiogenesis, regulating the epithelial-to-mesenchymal transition (EMT), and altering cell–cell and cell–ECM interactions ,40,41,42ADAMS  and ADAMTS  are a related family of metalloproteinases that, like MMPs, are involved in proteolytic processing, cell adhesion, integrin signaling, and ectodomain shedding. Several studies have indicated their role in cancer progression by regulating the extracellular microenvironment and immune evasion ,47. PartCollagens are the major components of the ECM that are secreted by fibroblasts, cancer cells, and endothelial and immune cells, and their abnormal alignment and high density results in the tumor-specific ECM. This collagen-rich, tumor-specific ECM with increased stiffness is known to promote malignant transformation, intravasation, EMT, and metabolic adaptability and, thus, metastasis ,55. CollThus, the enrichment of these pro-tumor ECM remodelers in the highly invasive M1 cells is potentially responsible for inducing a metastasis-favorable microenvironment at the secondary sites, thereby promoting the colonization of the injected M1 cells. Furthermore, previous studies have shown the oncogenic potential of MMP2/9 in HR-NB cell lines in vitro ,59,60; tIn accordance with this gene signature, M1 cells and their derived variants showed resistance towards IFN-γ-induced phenotype changes in vitro, specifically with MHC-I expression, potentially imparting them with the ability to evade immune surveillance in immunocompetent mice. IFN-γ is known to induce an anti-tumor response by enhancing the expression of MHC class I molecules in cancer- and antigen-presenting cells, thereby facilitating the presentation of tumor cells in T-cell-mediated killing . AdditioTaken together, in vitro characterization and in vivo validation, along with the RNA-seq analysis, reveal the potential mechanisms/pathways that may underly the specifically acquired metastatic ability of the newly developed cell lines. Additionally, we have previously demonstrated extracellular vesicles as one of the mechanisms responsible for the metastatic nature of M1 cells by inducing a pre-metastatic niche at secondary sites . With thDue to its highly metastatic nature, chemotherapy resistance, and low immunogenicity, HR-NB is among the leading causes of cancer-related deaths in pediatric patients. Despite treatment with aggressive multi-modal radio-chemo-immunotherapy, most patients with metastatic HR-NB develop refractory or relapsed disease, resulting in significantly lower overall survival . Importa