FOXA2 haploinsufficiency (FOXA2+/− iPSCs) followed by beta-cell differentiation to understand the role of FOXA2 during pancreatic beta-cell development. Our results showed that FOXA2 haploinsufficiency resulted in aberrant expression of genes essential for the differentiation and proper functioning of beta cells. At pancreatic progenitor (PP2) and endocrine progenitor (EPs) stages, transcriptome analysis showed downregulation in genes associated with pancreatic development and diabetes and upregulation in genes associated with nervous system development and WNT signaling pathway. Knockout of FOXA2 in control iPSCs (FOXA2−/− iPSCs) led to severe phenotypes in EPs and beta-cell stages. The expression of NGN3 and its downstream targets at EPs as well as INSUILIN and GLUCAGON at the beta-cell stage, were almost absent in the cells derived from FOXA2−/− iPSCs. These findings indicate that FOXA2 is crucial for human pancreatic endocrine development and its defect may lead to diabetes based on FOXA2 dosage.FOXA2 has been identified as an essential factor for pancreas development and emerging evidence supports an association between FOXA2 and diabetes. Although the role of FOXA2 during pancreatic development is well-studied in animal models, its role during human islet cell development remains unclear. Here, we generated induced pluripotent stem cells (iPSCs) from a patient with  Monogenic diabetes (MD) is caused by a mutation or defect in a single gene-regulating beta-cell development and/or function3. Several heterozygous mutations in the TFs expressed during pancreatic development are associated with a specific form of MD, known as MODY. However, homozygous mutations of the same TFs lead to neonatal diabetes, which can be associated with pancreatic hypoplasia/agenesis in some mutations4. This indicates that the onset and severity of the diabetes phenotype are correlated with the dosage of the TF expression during pancreatic development.During human development, early endodermal tissue becomes specified toward a pancreatic fate before evagination of pancreatic buds, populated with pancreatic progenitors. All adult pancreatic cells are originated from the same progenitors expressing a group of transcription factors (TFs), including PDX1, SOX9, FOXA2, NKX6.1, HNF6, and PTF1A5. Foxa2 knockout mice die at an early embryonic stage and show developmental defects in the foregut (FG) and neural tube8. During pancreatic development, FOXA2 is expressed at early stages starting from the endoderm stage and its protein level is increased during the endocrine specification stage9, while the exocrine and ductal cells express a low level of FOXA29. Mouse studies showed that Foxa2 is important for islet development and beta-cell functionality12 and its specific deletion in beta-cells leads to hyperinsulinemic hypoglycemic phenotype14. In human, previous reports demonstrated that patients with heterozygous FOXA2 mutations develop hyperinsulinemia, hypoglycemia, hypopituitarism, endodermal organ defects, and craniofacial abnormalities16. Recent genomic studies found that type 2 diabetes (T2D) risk alleles are associated with FOXA2-bound enhancers in human17. Another recent study reported a patient with diabetes due to a heterozygous missense variant in FOXA2, indicating that FOXA2 defect can lead to MD18. FOXA2 is known to regulate the expression of multiple TFs controlling pancreatic endocrine cell fate and insulin secretion20.FOXA2 is expressed in several tissues and performs distinct functions as evident in the phenotypes of mouse models21, which do not fully reflect human phenotypes. Recent progress in the induced pluripotent stem cell (iPSC) technology allowed us to generate in vitro models to study human genetic diseases22. To our knowledge, there is only one recent study that used FOXA2 knockout hESCs (FOXA2−/− hESCs) to study FOXA2 during pancreatic progenitor differentiation23. To better understand the role of FOXA2 in beta cell development and diabetes progression, we established iPSCs from a patient with a heterozygous deletion in FOXA2 (FOXA2+/− iPSCs)24. We showed a number of key genes essential for pancreatic development were dysregulated in FOXA2+/− iPSC-derived pancreatic cells and the results obtained were further validated by generating CRISPR/Cas9-mediated FOXA2 knockout (FOXA2−/− iPSCs). Our results suggest that FOXA2 have a crucial role in pancreatic endocrine development in human.Most of the information available on FOXA2 functions have been obtained from animal studiesFOXA2 gene 24 and from a healthy control 25. In this study, we generated additional iPSC lines from another healthy control (Ctr2-iPSCs). All the established iPSC lines showed the expression of the pluripotency markers and showed the ability to differentiate into the three germ layers  ] and a deletion  as expected24 and as reported in the patient sample26  and endocrine progenitors (EPs). FOXA2 protein and mRNA levels were markedly reduced in PP2 derived from FOXA2+/− iPSCs , HNF1A, and PAX4 as well as the exocrine precursor markers, PTF1A and AMYLASE. Interestingly, all these pancreatic markers were significantly downregulated in PP2 derived from FOXA2+/− iPSC lines compared to those derived from Ctr-iPSCs  and 427 upregulated  differentially expressed genes (DEGs) in PP2 derived from FOXA2+/− iPSCs compared to those generated from Ctr-iPSCs  and 564 upregulated  DEGs in EPs derived from FOXA2+/− iPSCs compared to those generated from Ctr-iPSCs + cells. However, the expression levels of INS, NKX6.1, C-PEP, and GLUCAGON (GCG) were downregulated as examined by immunostaining and flow cytometry in stage 7 derived from FOXA2+/− iPSCs compared to those derived from Ctr-iPSCs  and KCl stimulation compared to low glucose levels  and high (20 mM) glucose and KCl stimulation was measured. Beta cells derived from FOXA2els Fig. . We did SCs Fig. . HoweverSCs Fig. . These rFOXA2+/− iPSCs and to exclude the possibility of cell line variations, we used CRISPR/Cas9 to KO the FOXA2 gene in the iPSCs (FOXA2−/− iPSCs)  controls. Characterization of both FOXA2−/− iPSC lines showed that they were pluripotent and displayed normal karyotype  derived from FOXA2+/− iPSCs and FOXA2−/− iPSCs  at the PP2 stage on reversing the phenotype associated with FOXA2 deficiency  with hypoglycemic hyperinsulinemic phenotype due to two different FOXA2 haploinsufficiency carrying the genetic information of the patient. Our results indicate that FOXA2 haploinsufficiency negatively impacted endocrine islet development by downregulating key TFs associated with islet cell development and upregulating genes associated with neuronal development and WNT and BMP pathways. Taken together with other recent reports23, our findings suggest that FOXA2 defects may lead to monogenic diabetes based on the gene dosage. However, it will be of interest to perform further studies to investigate how FOXA2 works with other TFs to main normal pancreatic endocrine development in human.In conclusion, our current study provides the first human iPSC model for The protocols to obtain blood samples were approved by the Institutional Review Board (IRB) of Sidra Medicine (no. 1702007608) and QBRI/HBKU (no. 2018-002). Informed consent forms were signed by human subjects.FOXA2 heterozygous deletion  and healthy controls (Ctr1-iPSCs and Ctr2-iPSCs) as we previously reported25. All lines were extensively characterized as previously reported25. The first four stages of the pancreatic differentiation were performed using our protocol27 and for further differentiation to beta cells, Rezania et al.28 protocol was used  for 4 days during stage 4 and the control cells were treated with DMSO. For BMP inhibition, the cells were treated every day during stage 4 with 100 ng/mL NOGGIN, while the control cells were treated with 50 ng/mL of NOGGIN. The cells were collected at end of stage 4 to examine the effect of WNT and BMP inhibition on the dysregulated genes associated with 29. RT-PCR and qPCR were performed as previously reported25.  experiments and data analysis were performed as we previously reportedFOXA2 iPSC lines from two control iPSC lines (Ctr1 and Ctr2). The cells were transfected with a plasmid expressing spCas9 and gRNA  using Lipofectamine Stem Transfection Reagent following the manufacturer’s instructions (ThermoFisher Scientific)  and high (20 mM) glucose, which was repeated two times. In the end, the clusters were incubated in low glucose with 30 mM KCL to depolarize the cells and release their C-PEP (C-PEPTIDE) contents. The C-PEP concentration in the collected supernatants was measured using Human C-Peptide ELISA Kit . Each value of C-PEP level of low or high glucose condition was normalized to its own total content of C-PEP in each sample. The stimulation indices were determined through the ratio between the C-PEP level at high glucose concentration to its own basal level secretion at the two challenges.At the end of day 2 of stage 4, the cells were dissociated with TrypLE and resuspended in the differentiation medium with 10 μM Y-27632. The resuspended cells were either transfected with FOXA2 plasmid , PDX1 plasmid , or the empty vectors. Transfection was carried out using the Lipofectamine™ 3000 Transfection Reagent following the manufacturer’s instruction . At 48 h post-transfection (end of stage 4), the cells were harvested for RNA and protein extraction. Some experiments were continued until the end of stage 7 (beta-cell stage) for further analyses.t-test by Prism 8.At least three biological replicates were used in most of the experiments and statistical analysis was carried out using unpaired two-tailed Student’s Supplementary Table 1: Differentiation MediaSupplementary Table 2:Antibody detailsSupplementary Table 3: Primer listSupplementary Table 4: Top downregulated genes in PP2 derived from FOXA2+/- iPSCs in comparison to those derived from Ctr-iPSCsSupplementary Table 5: Top Upregulated genes in PP2 derived from FOXA2+/- iPSCs in comparison to those derived from Ctr-iPSCsSupplementary Table 6: Top downregulated genes in the EPs derived from FOXA2+/- iPSCs in comparison to those derived from Ctr-iPSCsSupplementary Table 7: Top upregulated genes in the EPs derived from FOXA2+/- iPSCs in comparison to those derived from Ctr-iPSCsSupplementary Table 8: gRNA sequences for FOXA2Supplementary Figure 1Supplementary Figure 2Supplementary Figure 3Supplementary Figure 4Supplementary Figure 5Supplementary Figure 6


Retroelements (REs) occupy a significant part of all eukaryotic genomes including humans. The majority of retroelements in the human genome are inactive and unable to retrotranspose. Dozens of active copies are repressed in most normal tissues by various cellular mechanisms. These copies can become active in normal germline and brain tissues or in cancer, leading to new retroposition events. The consequences of such events and their role in normal cell functioning and carcinogenesis are not yet fully understood. If new insertions occur in a small portion of cells they can be found only with the use of specific methods based on RE enrichment and high-throughput sequencing. The downside of the high sensitivity of such methods is the presence of various artifacts imitating real insertions, which in many cases cannot be validated due to lack of the initial template DNA. For this reason, adequate assessment of rare (< 1%) subclonal cancer specific RE insertions is complicated.Here we describe a new copy-capture technique which we implemented in a method called SeqURE for Sequencing Unknown of Retroposition Events that allows for efficient and reliable identification of new genomic RE insertions. The method is based on the capture of copies of target molecules (copy-capture), selective amplification and sequencing of genomic regions adjacent to active RE insertions from both sides. Importantly, the template genomic DNA remains intact and can be used for validation experiments. In addition, we applied a novel system for testing method sensitivity and precisely showed the ability of the developed method to reliably detect insertions present in 1 out of 100 cells and a substantial portion of insertions present in 1 out of 1000 cells. Using advantages of the method we showed the absence of somatic Alu insertions in colorectal cancer samples bearing tumor-specific L1HS insertions.This study presents the first description and implementation of the copy-capture technique and provides the first methodological basis for the quantitative assessment of RE insertions present in a small portion of cells.The online version contains supplementary material available at 10.1186/s13100-020-00228-6. Retroelements (REs) in the human genome comprise more than 1.5 million copies that arise as a result of a process called retrotransposition. Three major classes of REs are LTR retroposons, LINEs, and SINEs that include the primate-specific Alu family. Only a tiny portion of REs is still capable of retrotransposition including more than one hundred of autonomous LINE-1 (L1) copies , 3 and aThe method is based on consecutive selective amplification of both 5′ and 3′ flanking regions adjacent to the copies of retroelements belonging to young RE subfamily of interest Fig. . For detAt the next step fragmented DNA is ligated to stem-loop DNA adapters containing Unique Molecular Identifiers (UMI) . These rNext, the biotinylated primer specific to AluYa5 (or AluYb8) fragment containing 3 diagnostic mutations for Ya5 (or specific 7 nt fragment for Yb8) is used for linear amplification of retroelement 5′ flanks. The biotinylated primer along with the biotinylated dCTP is used to introduce biotin into generated ssDNA molecules. After linear amplification the obtained product is denatured, the biotinylated 5′ flank copies are captured and removed from the reaction with the use of streptavidin-coated magnetic beads. Importantly, the restricted and adaptor-ligated genomic DNA template remains intact. This DNA is used for the second linear amplification with the primer complementary to the same region characteristic for AluYa5 (or AluYb8) but in the opposite direction (towards the 3′ flank of the insertion). This product is also captured by streptavidin-coated magnetic beads. The remaining initial template DNA is used to confirm insertions by locus-specific PCR. Captured linear DNA is amplified in the subsequent PCR to generate libraries of AluYa5 (AluYb8) flanks (separately for 5′ and 3′ flank). The second PCR is used for sample barcoding and generates libraries ready for sequencing on Illumina. The resulting fragments consist of a short part of the Alu repeat, adjacent genomic flank (5′ or 3′), a part of the adapter with UMI, and standard parts necessary for Illumina sequencing including sample barcodes. 3′ flanking library also includes the polyA tail of the Alu.After sequencing, we use a custom computational pipeline  to map ahttp://dbrip.brocku.ca/) or 1000 genomes project databases [To evaluate method sensitivity, we used polymorphic Alu insertions. Using our method we first identified coordinates of AluYa5 insertions in the genomes of four healthy individuals . Then we compared these coordinates and identified in total 137 AluYa5 insertions that are absent in the genome of individual D1 and present in the genome of one of the individuals D2, D3, or D4 , 37 out of 45 Alu for individual D3 (0.3%) and 36 out of 48 Alu for individual D4 (0.1%) have nearest 3′ restriction site in the range of 30–529 bp (same as for the 5’flank). Next, we searched the 3’flank library for the insertions that are present in both 5′ and 3′ flank libraries. As a result, we were able to find 32–34 out of 36 insertions present in 1% (D2) of cells, 26–29 out of 37 insertions present in 0.3% (D3) of cells and 16–18 out of 36 insertions present in 0.1% (D4) cells  of insertions present in 1% of the cells in a sample can still be found with sequencing depth of 2 million reads. As expected, for the cells with 10 times lower concentration (0.1%) even 15 million of reads is not enough to catch 100% of the insertions, however, 28 out of 48 (approximately 60%) are repeatedly found in both replicates. Around 20% of insertions are still repeatedly detectable with sequencing depth of 2 million in both replicates . We plotted normalized UMI count in replicate 1 versus UMI count in replicate 2 for each of the identified Alu insertion from individuals D1-D4  that left after two rounds (5′ and 3′ flank) of linear amplification and capture was used as a template for locus-specific PCR. We selected 10 non-reference Alu insertions which have a distance between 5′ and 3′ flanks restriction sites in the range 400–600 bp. Next, we removed short (100–300 bp) template DNA molecules from the mixture by magnetic beads. This procedure depletes DNA fragments that do not contain Alu insertions  originating from the genome of individual D1. After that, we performed locus-specific PCR with the primers corresponding to unique flanking regions of each insertion. We observed PCR product of expected length for 4 out of 4 insertions present in the genome of D2 (1% of cells), for 3 of 4 insertions present in the genome of D3 (0.3% of cells) and for 1 of 2 insertions present in the genome of D4 (0.1% of cells). Obtained PCR products were sequenced by Sanger method which in all cases allowed to confirm the insertions by the alternative method and were able to identify TSD indicating true Alu insertions.Using our method, we performed a search for cancer-specific Alu Ya5 and AluYb8 insertions in 6 paired  colorectal cancer samples. Libraries of 5′ and 3′ flanks were prepared using SeqURE method and sequenced. For each 5′ flank library we obtained a minimum of 2,173,258 reads and the difference between tumor and normal samples in each pair was less than 2-fold. After data processing and artifact filtration, we compared lists of Alu insertion coordinates in paired normal and tumor samples. Only the insertions with 2 or more different UMI present in the tumor sample and absent in corresponding paired normal sample were considered tumor-specific candidates. We identified 10 potential tumor-specific Alu insertions in the 5′ flank libraries of 5 out of 6 tumor samples (see Table n = 3) we failed to get PCR product. The obtained PCR products for 4 tumor-specific insertions were sequenced by Sanger method. In all cases we confirmed tumor-specific L1HS insertions.To further proof the developed copy-capture approach we identified cancer-specific insertions of another family – L1HS in the same set of paired tumor/normal samples. As amplification and sequencing of 5’flanking regions of L1 insertions is yet challenging we generated only the library of 3′ flanks, using the same experimental approach as for Alu 3′ flank libraries. Genomic DNA was digested with another pair of restriction enzymes and ligated to adapted with the same pan-handle like structure (see methods for details). After linear amplification with L1HS specific biotinylated primer 3′ flank copies were extracted with streptavidin-coated magnetic beads and amplified in the subsequent 2 round PCR to generate ready-for-sequencing sample-barcoded libraries. Same as for Alu libraries, template DNA stays intact and is used for validation by locus-specific PCR. For each library we obtained a minimum of 219,970 reads and the difference between tumor and normal samples in each pair was less than 2-fold. In six paired samples combined we found 1119 L1HS insertions including 854 reference insertions (hg38), 188 previously known non-reference insertions  and 77 In the recent years it is becoming widely accepted that RE transcriptional and transpositional activity can play a significant role in cancer. New insertions can disrupt cellular genes  either by direct integration into a gene , 29 or vWe use a modified selective amplification approach , 31 to gThe significant drawback of this approach is the inability to identify TSD – the hallmark of the retrotransposition event. As a result, even with very high sensitivity and vigorous artifact filtration pipelines, many potential insertions are not confirmed by the locus-specific PCR. This limitation can be overcome by the amplification of both flanking regions (5′ and 3′). However, this approach is not reliable when detecting insertions present in a minor fraction of cells if different aliquots of DNA from the same sample are used for library preparation and validation. Here we consecutively amplify both adjacent genomic regions of each insertion from the same template which significantly increases the chance to retrieve both flanks and identify TSD. In addition, sequence of one flank is used to validate the insertion found in the library of another flank: when we know the insertion coordinate from one library we can predict the length of another flank as we use restrictase for DNA fragmentation. If the corresponding sequence has suitable length for amplification and sequencing but not found in another flank library – this indicates false insertion. Moreover, template genomic DNA used for libraries preparation stays intact and can be used for locus-specific PCR to confirm insertions. Using spike-in controls we show that this approach can indeed produce reliable results. Most of the insertions that have corresponding sequences in 5′ and 3′ flank libraries were confirmed by locus-specific PCR from the same DNA template. We also directly characterized the sensitivity of the method. We show that the majority of insertions present in as little as in 1% of cells can be reliably (in two replicates) identified with the sequencing depth of only 2 million of 150 + 150 paired reads (of 0.6 Gb) per sample. This corresponds to 0.2x human genome coverage. It should be mentioned that uneven amplification efficiency of different flanking sequences results in different sequencing coverage for each particular RE insertion. As it was shown previously  amplificUnlike L1 insertions, tumor-specific Alu insertions are rare in cancers including colorectal cancer . As a prTo further validate the method, we searched for tumor specific L1HS insertions in the same set of samples using the same methodological approach. We identified 10 candidate insertions with UMI counts ranging from 2 to 16 and fully confirmed 4 of them by orthogonal approach. These results indicate successful application of our method on real samples, and at the same time, demonstrate the necessity for sequencing of both flanks for reliable tumor-specific RE identification. Additionally, the identified tumor specific L1HS insertions indicate that at least 3 out of 6 analyzed colorectal tumor samples were subjected to retropositional activity. At the same time, we accurately showed absence of tumor specific Alu insertions in all 6 samples. Taken together, these facts indicate that Alu insertions in cancer occur much rarer than L1 and active L1 machinery alone is insufficient for Alu retropositional activity.Most of the previously described techniques for targeted RE capture like RC-seq  use origSix colorectal cancer samples were obtained during R0 partial colectomy from patients with stage II or III colorectal adenocarcinoma (5 patients) or carcinoma in situ (1 patient) treated at the A.N. Ryzhikh National Medical Research Centre for Coloproctology. The study was approved by the local ethical committee and all the patients gave standard informed consent. Tumor and normal tissue fragments were taken under the supervision of a pathomorphologist in the shortest possible time after intestinal resection (not more than 30 min). DNA was extracted with QIAamp DNA Mini Kit (Qiagen) according to manufacturer’s protocol.PBMC of four healthy donors were isolated from peripheral blood by standard Ficoll-Paque  centrifugation protocol. T-cells from different individuals were stained by the following antibodies: individual D2 CD3-eFluor450 , individual D3 - CD3-FITC , individual D4 CD3-PC5 . Cells of individual 1 were not stained. FACS sorting was performed in 5 replicates by BD FACS Aria III. Cells were gated based on side and forward scatter. Each cells mixture contained 50,000 cells of individual D1, 500 cells of individual D2, 150 cells of individual D3 and 50 cells of individual D4. After sorting three out of five aliquots were resorted to evaluate the resulting number of cells in the mixture. DNA from sorted cells was isolated using QIAGEN DNeasy Blood and Tissue Kit (Qiagen).To avoid possible contamination, we first prepared libraries from cell mixtures and sequenced them. After that we made libraries from DNA of individuals D1-D4 separately to identify individual-specific Alu insertions present in the genome of individuals D2, D3 or D4 and absent in the genome of individual D1.Thirty ng of genomic DNA was digested by incubation in 10 μl of 1x FD buffer with 5 U of FspBI and 5 U of Csp6I  for 30 min at 37 °C. For adapters ligation fragmented DNA was diluted by 20 μl of 1x FD Buffer with 20 μmol of ATP (Thermo Fisher Scientific), 50 pmol of SL-adapter  for 30 min at 37 °C and 30 min at 65 °C. For adapters ligation fragmented DNA was diluted by 20 μl of 1x FD Buffer with 20 μmol of ATP (Thermo Fisher Scientific), 50 pmol of SL-adapter-2  and used for the 2nd amplification to obtain DNA fragments of opposite (5′) L1HS flanks. The amplification profile, capture and purification condition were the same as for 3′- flank libraries. The captured amplicons (copies) were indexed at the same condition as 3′ flanks amplicons, purified with AmPure XP beads, mixed equimolarly and sequenced on Illumina MiSeq paired end 150 + 150.Raw sequencing reads were processed as described previously . As a rePrimers for the specific genomic regions were designed with primer-blast and GeneRunner programs. Twenty-five μl PCR reactions containing 200 μM of each of dNTP, 0.2 μM of each of forward and reverse locus-specific primers (Additional file Additional file 1: Supplementary Table 1. A detailed description of individual-specific Alu insertions.Additional file 2: Supplementary Table 2. TSD identification.Additional file 3: Supplementary Table 3. Oligonucleotide sequences used for library preparation and locus-specific PCR.


The sirtuin (SIRT) proteins are a highly conserved family of NAD+-dependent deacetylases that regulate histones as well as non-histone proteins. Seven sirtuin genes have been identified (SIRT1 to SIRT7) in mammals. SIRT1, SIRT6, and SIRT7 are primarily localized in the nucleus. SIRT2 is localized mainly in the cytoplasm. SIRT3, SIRT4, and SIRT5 are often located in the mitochondria. Therefore, the sirtuin family proteins exert their diverse functions at various cellular locations and regulate metabolism, stress responses, growth and aging processes. The sirtuin proteins are often considered as nutrient sensors. This study assessed the expression of sirtuin genes in C2C12 muscle cells under glucose stress conditions at different time points. Expression of all seven sirtuins was confirmed by Real-Time PCR analysis. SIRT1 (24 h) and SIRT3 (6 h and 15 h) are highly expressed under low glucose (2.7 mM) and high glucose (25 mM) conditions, whereas SIRT2 and SIRT4, SIRT5, SIRT6 and SIRT7 expressions were either relatively lower or there was no significant change under glucose stress conditions. Our results indicate that SIRT1 and SIRT3 demonstrated the greatest fluctuation in response to glucose stress, whether high or low glucose. These findings will help elucidate the role of sirtuins in the regulation of cellular processes, including metabolism. It will also help to enhance our understanding of the roles of sirtuin genes in regulation of blood sugar fluctuation in normal persons and diabetic patients, as well as in elderly individuals, many of whom are insulin resistant and “prediabetic” or diabetic.


C-C motif ligand 2 (CCL2) was originally reported as a chemical mediator attracting mononuclear cells to inflammatory tissue. Many studies have reported that CCL2 can directly activate cancer cells through a variety of mechanisms. CCL2 can also promote cancer progression indirectly through increasing the recruitment of tumor-associated macrophages into the tumor microenvironment. The role of CCL2 in cancer progression has gradually been understood, and various preclinical cancer models elucidate that CCL2 and its receptor C-C chemokine receptor 2 (CCR2) are attractive targets for intervention in cancer development. However, clinically available drugs that regulate the CCL2–CCR2 axis as anticancer agents are not available at this time. The complete elucidation of not only the oncological but also the physiological functions of the CCL2–CCR2 axis is required for achieving a satisfactory effect of the CCL2–CCR2 axis-targeted therapy. C-C motif ligand 2 (CCL2) was discovered first among CC chemokines in 1989 and helped to propel subsequent discoveries of other CC chemokines . The bioThere is a number of studies suggesting that CCL2 can activate cancer cells through a variety of mechanisms. Initially, CCL2 was focused on and studied as a key molecule in the migration and proliferation of prostate cancer cells . CCL2 diAlthough a great number of the studies focusing on the function of CCL2 in cancer progression used prostate and breast cancer cells, more recent CCL2 studies have incorporated colorectal, lung, gynecological, gastric, and other types of cancer cells. Akt activation by phosphatidylinositol phosphate kinase gamma (PIPKIγ)-mediated STAT3 phosphorylation induces CCL2 expression in colorectal cancer cells and silencing PIPKIγ greatly decreases CCL2 expression at both the mRNA and protein levels, leading to reduced chemotaxis of cancer cells to macrophages . In metaSince CCL2 is also known by the alias monocyte chemoattractant protein-1 and activates macrophages, inactivation of cancer cells by the inhibition of the CCL2–CCR2 axis are partly presumed to be caused by the reduction of tumor-associated macrophages (TAMs) at the primary and metastatic site, inducing the destruction of the immunosuppressive tumor microenvironment and the susceptibility to the antitumor T lymphocyte response. TAMs play a major role in cancer progression by affecting diverse processes such as immunosuppression, angiogenesis, and tumor cell proliferation and metastasis during cancer progression ,41,42. IMyeloid-derived suppressor cells (MDSCs) also foster cancer cell activity by suppression of T lymphocytes and natural killer cells, while simultaneously they can recruit regulatory T lymphocytes to further promote immunosuppression . DeletioCAFs are stromal components and play an important role in tumor progression by regulating the tumor microenvironment and primarily releasing proteolytic enzymes, growth factors, and cytokines ,59. PancConsistent with the role of CCL2 in cancer progression, serum CCL2 levels and CCL2 expression levels in tumor tissue have been reported as potential biomarkers in a variety of cancer patients .The usefulness of serum CCL2 in prostate cancer patients was supported by a study using the serum samples of 255 and 124 men with and without prostate cancer, respectively, which reported that serum CCL2 levels in men with prostate cancer were significantly higher than those without. In addition, among the 255 patients with prostate cancer, those with serum CCL2 level ≥ 320 pg/mL had significantly poorer overall survival and prostate cancer-specific survival, higher TNM stages, and worse histological grade than those with serum CCL2 level < 320 pg/mL, indicating the potential of serum CCL2 level as a prognostic biomarker as well . The outWhen the preoperative serum CCL2 concentration of 45 colorectal cancer patients was measured, the 5-year survival rate was significantly better in the group with low preoperative CCL2 level than in the group with high preoperative CCL2 level . In a stSerum CCL2 was significantly higher in HCC patients in a study that measured CCL2 levels from serum samples from 98 resectable HCCs, 101 patients with chronic hepatitis B, and 100 asymptomatic hepatitis B and C virus carriers. In addition, the serum CCL2 also correlated with the disease stage of HCC and the combination of alpha-fetoprotein and CCL2 has significantly superior discriminative ability than alpha-fetoprotein alone . In examSerum CCL2 levels in 135 breast cancer patients were measured and elevated and serum CCL2 levels were significantly correlated with advanced cancer stage and lymph node metastasis . ImmunohA study of 212 pancreatic cancer patients found that CCL2 serum levels in pancreatic cancer patients were significantly higher than in normal healthy subjects . In a stCCL2 and CCR2 expression was analyzed by immunohistochemical staining and its correlations with clinicopathologic features and prognosis were evaluated in 221 patients with diffuse large B-cell lymphoma, and high expression of CCL2 or CCR2 was correlated with clinicopathological characteristics and indicated significantly poorer overall survival and progression-free survival . In a stAs a result of examining the peripheral blood of 60 patients with gastric cancer and 20 healthy subjects, preoperative serum levels of CCL2 in patients with gastric cancer were significantly higher than those in healthy controls. In addition, preoperative serum CCL2 was significantly correlated with lymph node metastasis . A studyIn a retrospective study including 86 ovarian cancer patients, 67 benign ovarian cysts, and 42 healthy women, ovarian cancer patients had significantly higher serum CCL2 levels, and serum CCL2 levels were statistically significantly correlated with histological malignancy . In a stComparing 142 lung cancer patients with 141 non-lung cancer subjects, CCL2 was significantly elevated in lung cancer patients . WesternExamining CCL2 levels in urine samples from 60 bladder cancer patients and 20 control healthy subjects revealed CCL2 levels in the urine of patients with bladder cancer correlated significantly with TNM stage and cancer grade . ImmunohExamination of serum CCL2 and TNF-α in 297 patients with pretreatment laryngeal cancer showed that CCL2 and TNF-α levels were independent predictors of overall survival and distant metastasis-free survival . ImmunohThe effects of the orally administered CCR2 inhibitor PF-04136309 in combination with leucovorin (folinic acid), fluorouracil, irinotecan, and oxaliplatin (FOLFIRINOX) in patients with treatment-naive locally advanced pancreatic ductal adenocarcinoma were assessed in a phase Ib study; ultimately, 16 (49%) of 33 patients receiving FOLFIRINOX plus PF-04136309 achieved an objective tumor response, with local tumor control achieved in 32 (97%) patients . HoweverLosartan, a type I angiotensin II receptor antagonist, is reported to have the potential to act as direct CCR2 antagonists. Losartan and its primary metabolite, EXP-3174, inhibit CCL2-mediated monocyte recruitment to the metastatic site through the inhibition of CCL2-induced Erk1/2 activation and significantly reduce the metastatic burden in breast and colon cancer models . SimilarPreclinical studies using the mouse model were also reported. Inhibition of CCL2 improved radiation resistance using mice transplanted with pancreatic duct adenocarcinoma cell lines . In a prAlthough many studies have proven the key roles of the CCL2–CCR2 axis in tumor progression in a variety of cancers, there are no clinically available drugs that can modulate the CCL2–CCR2 axis as anticancer agents so far. As described earlier, a high serum CCL2 level may reflect the activity of cancer cells because CCL2 is produced by them; however, some studies have reported conflicting results that a low serum CCL2 level contributed to a worse prognosis in cancer patients . This diThe performance of the drug itself in exerting its potential effect to suppress cancer cell activity is also important to consider. Measurement of free CCL2 concentration following carlumab administration revealed a rapid fall in free CCL2 as expected; however, post-treatment free CCL2 concentrations rapidly rebounded and quickly exceeded pretreatment serum concentrations. These unexpected results suggest that carlumab was ineffective at suppressing free CCL2 concentration for a clinically meaningful period of time. As no apparent increase in CCL2 production rate was observed when the dose was increased from 1 to 50 mg/kg in cynomolgus monkeys, dose-escalation might be needed to adequately and constitutively suppress free CCL2 concentration . PF-0413The CCL2–CCR2 axis functions physiologically in various organs such as the lungs and digestive tract where a variety of immune cells accumulate. Blockade of the CCL2–CCR2 axis potentially causes unexpected effects in such organs. Although it is unclear whether each adverse event attributes to the blockade of the CCL2–CCR2 axis because PF-04136309 was used in combination with cytotoxic agents in a single-arm study, pulmonary toxicity and diarrhea as well as myelosuppression were reported as severe adverse events in clinical trials . AlthougIn summary, to achieve sufficient efficacy of CCL2–CCR2-targeted therapy for cancer patients, it is not sufficient to target cancer cells or immune cells alone and it is necessary to fully elucidate the physiological functions of the CCL2–CCR2 axis against a variety of normal cells expressing CCR2.


This study aims to identify the role of synovial fluid levels of a disintegrin and metalloproteinase with thrombospondin motifs 9 (ADAMTS9) and a disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS4) for the prediction of intraarticular steroid injection success in knee osteoarthritis (OA).A total of eighty-four advanced stage knee OA patients (42 with stage 3 OA and 42 with stage 4 OA) were enrolled in the study. Baseline and posttreatment outcomes were determined using Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC). Pretreatment synovial fluid ADAMTS9 and ADAMTS4 levels were measured by enzyme linked immunosorbent assay (ELISA). ‘’Total WOMAC score regression of 18% and above’’ was taken as a minimal clinically important difference (MCID) to indicate improvement. Determining the best predictors of intraarticular steroid injection success in both groups was evaluated by multiple logistic regression analyses.= 0.026). Decreasing synovial fluid ADAMTS9 levels (odds ratio (OR): 0.625, 95% confidence interval (CI): 0.437–0.893) were found to be predictive for the WOMAC score percent change ≥18 in all OA patients (P= 0.010). Decreasing ADAMTS9 levels in synovial fluid  were predictive for MCID in stage 3 OA patients (P = 0.039). Synovial fluid ADAMTS9 levels were significantly lower in the stage 4 OA group when compared with the stage 3 group. The level of synovial fluid ADAMTS9 was statistically significantly lower in the WOMAC score percent change ≥18% than the WOMAC score percent change <18% group in Stage 3 OA group (PThe lower levels of ADAMTS9 in synovial fluid may be used in conjunction with high WOMAC scores in the prediction of intraarticular steroid injection success and advanced stage knee OA patients. Knee osteoarthritis (OA) is a destructive joint disease in which joint manifestations include progressive loss of cartilage, cartilage calcification, osteophyte formation, and subchondral bone remodelling. Osteoarthritis is graded according to radiological findings of changes in the joints. The Kellgren and Lawrence (K&L) scoring system is used to denote the radiological stage in OA. In advanced stages of OA, progressive changes occur due to cartilage degradation [1]. The extracellular matrix (ECM) of cartilage consists of type 2 collagen and aggrecan, which is a significant proteoglycan [1]. Progressive changes in cartilage in OA are dependent on active cell processes mediated by cytokines and proteases [1]. The role of synovial inflammation in the progression of OA is well known [2]. Several studies demonstrated that inflammation of the synovial membrane played a role in the progress of joint degeneration by increasing the release of proteases, such as matrix metalloproteinases and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS), in the fibroblasts of cartilage . Members of the ADAMTS family, especially ADAMTS4 and ADAMTS9 are already known to bind and degrade ECM components and contribute to joint destruction processes in arthritis [5]. Zhang et al. [6] showed that ADAMTS4 levels in synovial fluid were significantly lower in late-stage OA than in early-stage OA. Peng et al. [7] reported that ADAMTS4 in synovial fluid might be a potential biomarker for cartilage degeneration. Previously, the aggrecanases ADAMTS4 and ADAMTS5 were shown to contribute to cartilage destruction and to be responsible for aggrecan degradation, especially in patients with early-stage OA [8]. Gok et al. [9] suggested that the cytosine adenine (CA) repeat polymorphism of the ADAMTS9 gene could be used to determine the radiological progression of OA.Although recent studies have already shown that ADAMTS4 and ADAMTS9 are involved in cartilage destruction in OA , the possible role of these matrix metalloproteinases in OA progression and their utility in the evaluation of treatment success remains unclear. This study aimed to determine the significance of synovial fluid ADAMTS4 and ADAMTS9 levels in OA progression, particularly in the assessment of the response to intraarticular steroid injection in advanced stage OA patients.Eighty-four advanced stage knee OA female patients (42 with stage 3 OA and 42 with stage 4 OA) with matching body mass index (BMI) were recruited consecutively from the outpatient clinic of Konya Beyşehir State Hospital in 2018. The diagnosis of knee OA was determined according to the radiographic features. The radiological stage was based on the K&L scale, which is accepted as a reference standard of the World Health Organization. It requires the presence of all five radiological criteria: osteophytes on the joint side, periarticular ossicles, joint area narrowing (JSN), small pseudocyst regions within the subchondral bone and a change in the formation of the bone ends. Inclusion criteria of OA patients: patients with radiological features  and stage 4  [12], women who have not received any intraarticular and/or systemic OA therapy before and a BMI between 30 kg/m2 and 18 kg/m2. Exclusion criteria: patients with other knee joint diseases , infectious diseases, septic arthritis, obesity, neurological or neuromuscular diseases, bone tumours, osteoporosis or trauma related fractures, diabetes mellitus, Addison’s disease, immune system disorder, rheumatological diseases and previous use of systemic steroids and intraarticular hyaluronic acid injections. Also, OA women who did not comply with stage 3 and stage 4 criteria, that is, no changes in X-ray or changes in only osteophyte or JSN, were not included in the study.All participants provided written informed consent, and the study ethics committee approval was obtained from the local Ethical Committee of the University . Clinical examination was performed, and anthropometric measurements, concomitant diseases  were recorded for all participants included in the study. Knee function was assessed before and 1 month after the treatment with intraarticular steroid injection, using the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC). The WOMAC score is composed of 24 parameters that include pain (score range: 0–20), stiffness (score range: 0–8), and functional impairment (score range: 0–68) [13]. Improvement in the WOMAC score after intraarticular injection was assessed within an average of one to 3 months. ‘’Total WOMAC score regression of 18% and above’’ was taken as a minimal clinically important difference (MCID) to indicate improvement [14].Only advanced stage OA patients who received an intraarticular injection of the steroid methylprednisolone acetate (40 mg to 1 mL) were included in the study. Patients managed with surgical treatment were excluded. Knee joint synovial fluid samples were obtained using sterile needles. The samples were stored at –80 °C until the day of analysis. Synovial fluid levels of ADAMTS4 and ADAMTS9 were analyzed using Eastbiopharm branded human ADAMTS4 and ADAMTS9 enzyme linked immunosorbent assay (ELISA) kit with an immunoassay device (Immulite 2000) and presented in pg/mL and ng/mL, respectively. The blood reference range of the ADAMTS9 assay kit is 5–400 ng/mL, and the blood reference range of the ADAMTS4 assay kit is 5–1000 pg/mL. The intraassay and interassay coefficients of variation of the kit were lower than 10% and 12%, respectively.Data analysis was performed using SPSS for Windows, version 22 . Whether the distributions of continuous variables were normal or not was determined using the Kolmogorov–Smirnov test, homogeneity of variances was evaluated using the Levene test. Continuous variables were shown as mean ± standard deviation (SD) or median (min–max), where applicable. While Student’s t-test compared the mean differences between groups, the Mann–Whitney U test was applied for comparisons of the median values. Patients with advanced OA, according to the K&L scale, were grouped into stage 3 and stage 4 OA. The optimal cutoff points of synovial fluid ADAMTS9 levels in advanced stage OA cases were evaluated by receiver operating characteristic (ROC) analyses, calculating the area under the curve (AUC) as giving the maximum sum of sensitivity and specificity for the variables test. Stage 3 and stage 4 groups were subgrouped into those with a WOMAC score percent change ≥18% and a WOMAC score percent change <18%. Synovial fluid ADAMTS9, synovial fluid ADAMTS4 levels and other variables were compared with Student’s t-test, responding and not responding to the improvement in WOMAC score in stage 3 and stage 4 groups. Also, categorical comparisons were performed using the χ2-test. Finally, multiple logistic regression analysis was performed separately in stage 3, stage 4, and all advanced stage knee OA patients to determine the effect of independent variables associated with response to intraarticular therapy. Adjusted odds ratios (ORs) and 95% confidence intervals (CI) were calculated for each variable. A P-value of less than 0.05 was considered statistically significant.n= 42; stage 4,n= 42) were enrolled in the study. Their baseline anthropometric and biochemical characteristics are given in Table 1. The mean age of the patients with stage 3 OA was 62.38 ± 6.78 years, and patients with stage 4 OA was 67.64 ± 5.63 years. The mean age of the patients with stage 4 OA was significantly higher than the stage 3 OA group (P< 0.001). There were no statistically significant differences among smokers in the groups (P= 0.766).A total of 84 BMI matched OA patients with advanced stage disease . WOMAC scores before intraarticular injection were 82.12 ± 6.18 and 65.21 ± 16.26. WOMAC scores after intraarticular injection were 67.26 ± 7.21 and 53.19 ± 12.72 respectively for stage 3 and stage 4 OA patients. WOMAC scores before and after intraarticular injection were significantly higher in the stage 3 OA group compared to the stage 4 OA group (P< 0.001and P < 0.001). Synovial fluid ADAMTS4 levels were 128.51 ± 27.79 pg/mL and 131,84 ± 35.47 pg/mL respectively in stage 3 and 4 OA groups. There were no statistically significant differences among synovial fluid ADAMTS4, WOMAC score change and concomitant diseases  between groups (Table 1).Synovial fluid ADAMTS9 levels were 5.92 ± 1.94 ng/mL in stage 3 OA and 5.02 ± 1.35 ng/mL in stage 4 OA cases . The patients with stage 4 OA had significantly lower levels of ADAMTS9 when compared with stage 3 OA group ; cutoff levels were determined, and AUC values were calculated. The AUC, best cutoff values, sensitivity, and specificity for distinguishing the groups for each parameter are given in Table 2. Synovial fluid ADAMTS9 levels were found to be statistically significant. When the groups that responded and did not respond to the treatment were compared within stage 3 and stage 4, the synovial fluid ADAMTS9 levels were 5.04 ± 1.44 ng/mL in the WOMAC score percent change ≥18% group, and 6.43 ± 2.01 ng/mL in the WOMAC score percent change <18% group.= 0.026). There were no statistically significant differences among age, smoking, synovial fluid ADAMTS4, and concomitant diseases between the groups in stage 3 (Table 3). Age, smoking, synovial fluid ADAMTS9, synovial fluid ADAMTS4, and concomitant diseases were not statistically different between WOMAC score percent change ≥18% and WOMAC score percent change <18% in stage 4 (Table 4).The level of synovial fluid ADAMTS9 was statistically significantly lower in the WOMAC score percent change ≥18% than the WOMAC score percent change <18% group in stage 3 OA (P= 0.010) (Table 5).At first, logistic regression analysis was performed to determine the variables associated with an 18% improvement in the WOMAC (MCID) of intraarticular steroid injection in all OA patients. Decreasing synovial fluid ADAMTS9 levels  were found to be predictive for the MCID in all OA patients  of intraarticular steroid injection treatment in stage 3 and stage 4 OA patients, respectively. Only decreasing ADAMTS9 levels in synovial fluid  were predictive for MCID in stage 3 OA patients (P = 0.039) (Table 6). Age, smoking, synovial fluid ADAMTS9, synovial fluid ADAMTS4, and concomitant diseases were not statistically associated with MCID in stage 4 (Table 6). Advanced stage OA is a chronic disease associated with progressive cartilage degeneration. Known triggers of OA include mechanical factors, age, and inflammation [15]. In recent years, neither systemic nor intraarticular medical treatment is effective in stopping the progression of OA [15]. Surgical treatment is the last resort in patients with OA. Several studies have focused on factors in the progression of OA in the elderly population. Kevorkian et al. [16] showed that the expression of ADAMTS1, ADAMTS5, ADAMTS9, and ADAMTS15 genes was significantly decreased and that the expression of ADAMTS16, ADAMTS2, ADAMTS14, and ADAMTS12 genes was increased in OA cartilage, compared to normal cartilage. Naito et al. [17] demonstrated that ADAMTS4 might play an important role in the degradation of aggrecan in human osteoarthritic cartilage. In our study, we found no significant differences in synovial fluid ADAMTS4 levels of stage 3 and stage 4 OA patients. However, the ADAMTS9 levels in the synovial fluid of stage 4 OA patients were significantly lower than those of stage 3 OA patients. Also, the WOMAC score in stage 4 OA group was significantly lower than in the stage 3 OA group. Pretreatment WOMAC scores were higher in the stage 3 OA group when compared with the stage 4 OA group, possibly pointing out the ongoing cartilage degeneration.In human chondrocyte cell lines, Coughlan et al. [10] showed that both ADAMTS5 and ADAMTS9 appeared to be important in cartilage ECM formation and turnover, and that depletion of ADAMTS5 and ADAMTS9 will lead to improved ECM formation and neocorticalization. Yaykasli et al. [11] demonstrated that leptin increased the expression of ADAMTS4, ADAMTS5, and ADAMTS9 genes and cartilage degeneration in human chondrocytes. Yang et al. [18] revealed abnormal increases in Cysteine-rich protein 61(Cyr61) and ADAMTS4 protein levels in OA tissues and chondrocytes. In the same study, Cyr61 reduced ADAMTS4 protein expression in chondrocytes, and Cyr61 interacted with ADAMTS4 to affect OA progression. Stanton et al. [19] found no significant phenotypic abnormalities in ADAMTS4 and ADAMTS5 knockout mice. Still, they found a significant reduction in the severity of surgically induced OA in ADAMTS5 knockout groups compared with ADAMTS4 knockout groups [19]. The present study demonstrates the prediction of intraarticular treatment success in stage 3 OA patients with synovial fluid ADAMTS9 levels. Intraarticular steroid injection was ineffective in stage 4 OA patients due to the severity of cartilage degeneration.In conclusion, we think that ADAMTS9’s low level of synovial fluid is significant in the stage 3 group where cartilage degeneration is continuing, and is meaningless in the stage 4 group where cartilage has wholly degenerated, there is no longer any extracellular matrix structure and there will be no regeneration. With this result, the significantly low level of ADAMTS9 may be related to the fact that intraarticular or systemic treatments will still be effective due to the ECM protease in the synovial fluid of stage 3 OA patients. The limitations of our study are mainly the lack of evaluation of other proteases and proteoglycans, which are risk factors of OA, a small number of patients and the controls had a history of joint injury and had been operated upon the knee for different reasons. The lack of a control group and early-stage OA is the limitation of the study. WOMAC 20,50, and 70 indexes could not be obtained due to the limited number of patients. The fact that no questionnaire related to the psychosocial status of patients has been conducted is another limitation of our study. Further prospective studies in larger cohorts are needed to validate the results of the present study. The authors would like to thank the staff at Beyşehir State Hospital and to all patients who participated in the study.


This study examined the prevalence of loneliness and relationship with cooking and eating habits among older adults using data collected in December 2019 from a nationally representative sample age 50-80 through the National Poll on Healthy Aging. Older adults who live alone were more likely those who lived with others (19% vs. 8%) and those with a high PHQ-2 score were more like than those with a low PHQ-2 score (20% vs. 11%) to report that they do not cook dinner most days (0-2 times/week). Those who rated their diet as fair/poor were more likely to indicate they cook few meals a week (0-2) compared to those who reported a good or very good/excellent diet (18% vs. 11 % vs 8%). Those with a high PHQ-2 score were less likely than those with a low PHQ-2 score to say they do major food shopping less than once a week (31% vs 47%). Those with a high PHQ-2 score were more likely to say that that always ate alone in the last week compared to those with a low PHQ-2 score (17% vs 7%). These findings demonstrate that older adults who lived alone, had a higher PHQ-2 score, and had a poorer diet were more likely to cook and do grocery shopping less often. Strategies and policies to support older adults to address depressive symptoms and to increase cooking and improve diet may have many health and social benefits and will be explored through this session.


IntroductionThis article is a retrospective analysis of the neurosurgical census at our institution to determine an optimal threshold for brain tissue oxygenation (PbtO2). The use of brain tissue oxygen monitoring has been in place for approximately three decades but data suggesting optimal thresholds to improve outcomes have been lacking. Though there are multiple modalities to monitor cerebral oxygenation, the monitoring of brain tissue oxygen tension has been deemed the gold standard. Still, it is not clear exactly how reductions in PbtO2 should be treated or what appropriate thresholds to treat might be. The aim of our study was to determine if our threshold of 28 mmHg for a good functional outcome could be correlated to the Glasgow Coma Scale (GCS) and Glasgow Outcome Scale (GOS).MethodsA retrospective analysis of the Arrowhead Regional Medical Center (ARMC) Neurosurgery Census was performed. Patients from 2017-2019 who had placement of Licox® cerebral oxygen monitoring sensors  were included in the analysis. Fifteen patients were consecutively identified, all of which presented with traumatic brain injury (TBI). Data on age, gender, days in the intensive care unit (ICU), days before discharge or end of medical care, admission GCS, hospital length of stay, GOS, maximum and minimum PbtO2 values for five days following insertion, minimum and maximum intracranial pressures (ICPs), and brain temperature were included for analysis. Patient data were separated into two groups; those with consistently higher PbtO2 scores  and those with inconsistent/lower PbtO2 scores . Standard student t-tests were used to find potential statistical differences between the groups (α = 0.05).ResultsThere were seven patients in the consistently high PbtO2 category (≥ 28 mmHg) and eight patients in the inconsistent/low PbtO2 category (<28 mmHg). The average maximum and minimum PbtO2 for the group displaying worse outcomes (as defined by GCS/GOS) was 23.0 mmHg and 14 mmHg, respectively. Those with consistent Day 2 PbtO2 scores of ≥ 28 mmHg had significantly higher GCS scores at discharge/end of medical care (p < 0.05). Average GCS for the patient group with >28 mmHg PbtO2 averaged over Days 2-5 group was 11.4 (n=7). Average GCS for the <28 group was 7.0 (n=8). The GCS for the >28 group was 63% higher than found in the <28 group (p = 0.03). GOS scores were significantly higher in those with consistently higher PbtO2 (≥ 28) than those with lower PbtO2 scores (< 28). The averages were 3.5 in the higher PbtO2 group as compared to 2 in the lower PbtO2 group.ConclusionAlong with ICP monitors and monitoring in the assessment of CPP, brain tissue oxygenation allows yet another metric by which to optimize treatment in TBI patients. At our institution, a PbtO2 level of ≥ 28 mmHg is targeted in order to facilitate a good functional outcome in TBI patients. Keeping patients at this level improves GCS and GOS at discharge/end of medical treatment. Given the observation that episodes of hypoxemia and ischemia following severe head injury increased mortality, the advent of brain tissue oxygenation monitoring in the 1990s had become an important tool in the intensive care unit (ICU) to avoid these insults . Along wThe Licox® brain tissue oxygenation system  was introduced into clinical practice in 1993, and initially saw most of its use in the setting of traumatic brain injury (TBI) and subarachnoid hemorrhage (SAH) [Though the use of brain tissue oxygen monitoring has been in place for close to three decades with improvement in outcomes, data suggesting optimal thresholds to change outcomes have been lacking -5. ThougPreviously, correlations between reduced brain tissue oxygenation and poor outcome in TBI patients have been observed . StudiesA retrospective analysis of the Arrowhead Regional Medical Center (ARMC) Neurosurgery Census was performed. Patients from 2017-2019 who had placement of Licox cerebral oxygen monitoring sensors were included in this analysis. Fifteen patients were consecutively identified, all of which presented with traumatic brain injury (TBI). Data on age, gender, days in ICU, days before discharge/end of medical care, admission GCS, GCS at discharge/end of medical care, GOS, maximum and minimum PbtO2 values for five days following insertion, minimum and maximum ICPs, and brain temperature were included. Patients were separated by those with consistently high post Day 2 PbtO2 (> 28 mmHg) measurements from those with inconsistent and/or lower post Day 2 PbtO2 scores (< 28 mmHg). Day 2 PbtO2 values were chosen in order to allow the Licox brain tissue monitoring device a period of calibration.We set the minimum requirement to be binned into the consistently high PbtO2 group as having at least ≥ 28 mmHg PbtO2 maximum for Days 3, 4, and 5 . This was the defined threshold below which to treat at our institution to attain a good functional outcome. Seven patients were identified with consistently higher PbtO2 scores (≥ 28 mmHg), and eight patients were identified with inconsistent/lower PbtO2 scores (< 28 mmHg). Standard t-tests were used to find potential statistical differences between the consistently high and inconsistently high PbtO2 groups. All statistical analysis, plot, and graph construction was done using Microsoft Excel (2016) with the Analysis ToolPak .The most common cause of TBI in our patient analysis occurred via motor vehicle accidents. Out of 15 patients, there were three deaths (20%). Our analysis included more men  than women , and most patients were within their first three decades of life. All patients were intubated, either in the pre-hospital setting or shortly after arrival, and were admitted to the ICU. Licox monitors were placed following initial stabilization typically within the first 24-48 hours of admission. PbtO2 levels were recorded hourly by nursing staff. Daily maximum and minimum values were obtained for the first five days of cerebral tissue oxygenation monitoring  were separated by those with consistently high post Day 2 PbtO2 (≥ 28 mmHg) measurements from those with inconsistent and/or lower post Day 2 PbtO2 scores (< 28 mmHg)  than those with lower PbtO2 scores (< 28 mmHg). The averages were 3.5 in the consistently high PbtO2 group compared to 2 in the inconsistently high/low PbtO2 group. The GOS for the >28 group was 62% higher than found in the <28 group (p = 0.01).External ventricular drain (EVD) length actually increases for those with consistently higher PbtO2. EVD length average is 20 days in the consistently high PbtO2 group as compared to 12 days in the inconsistent/low PbtO2 group. Those patients with consistently ≥ 28 mmHg PbtO2 scores had significantly longer hospital stays (average for ≥ 28 mmHg was 40 days compared with 12 days for those in the inconsistent/low PbtO2 group). Lastly, the consistently higher PbtO2 group had fewer deaths. Out of the 15 patients, three died, of which two had consistently low PbtO2 scores based on our threshold of 28 mmHg.Our threshold of 28 mmHg was also compared to 25 mmHg of PbtO2. We found that the length of EVD was not significantly longer . GOS is significantly higher in the higher PbtO2 (≥ 28 mmHg) group . ICU stay (days) was not significantly different . GCS was also found to be significantly higher in the 28 mmHg PbtO2 group . Lastly, we compared our threshold of 28 mmHg to 20 mmHg of PbtO2. We found that GOS was significantly higher in the higher PbtO2 (≥ 28 mmHg) group . GCS on discharge was significantly higher in the good group . ICU stay (days) was not significantly different . The length of time for EVD placement was significantly higher in the good group .Brain tissue oxygenation has become an important tool in the ICU to direct TBI patient interventions and to optimize treatment options. The management of TBI patients involves continued assessment and monitoring to prevent secondary injuries, such as hypotension and hypoxia after the initial irreversible primary brain injury has occurred. Prompt assessment of cerebral oxygenation is important because studies have shown that patients are most at risk for cerebral ischemia during the first few days following injury .At our institution, we consider a partial oxygen pressure above 20 mmHg to be indicative of a functional outcome and aim for readings above 28 mmHg for a good outcome . In thisIn our study, patients were separated by those with consistently high post Day 2 PbtO2 (> 28 mmHg) measurements from those with inconsistent and/or lower post Day 2 PbtO2 scores (< 28 mmHg) Table . We chosGOS was significantly higher in those with consistently higher PbtO2 (> 28 mmHg) than those with lower PbtO2 scores (< 28 mmHg). The average was 3.5 in the consistently high PbtO2 group as compared to 2 in the inconsistently high/low PbtO2 group. GOS for the >28 group was 62% higher than found in the < 28 group (p = 0.01).Controversy still exists as to the best placement of brain tissue oxygen monitor probes. Uninjured brain, injured brain, and penumbra surrounding the injured brain have all been advocated for, in regard to the placement of the probe. Some authors have waited two hours to record the first PbtO2 levels to allow the calibration of probes. Placement in all 15 patients was within the white matter of the frontal lobe in an uninjured brain. The Licox system is able to measure both oxygen and temperature in the intracranial cavity, with the intention of providing additional metrics to inform clinical decision-making. Potential interventions include the augmentation of the ventilator settings, augmentation in CPP, and the addition of sedation . The potThe consistently higher PbtO2 group had fewer deaths. Out of the 15 patients, three died, of which two had consistently low PbtO2 scores, based on our threshold of 28 mmHg. This was in line with previous studies with a threshold similar to ours for PbtO2 levels. Erikkson et al. found that PbtO2 levels below 29 mmHg in the first 72 hours predicted increased mortality in TBI patients . Though We did find that EVD length and hospital stay actually increases for those with consistently higher PbtO2. EVD length average was 20 days in the consistently high PbtO2 group compared to 12 days in the inconsistent/low PbtO2 group. Those patients with consistently high PbtO2 scores had significantly longer stays with an average of 40 days as compared with 12 days for those in the inconsistent/low PbtO2 group. We hypothesize that this increased length is a result of both increased mortality in the inconsistent or low PbtO2 groups, as well as more aggressive care in those patients who were deemed to have a better prognosis.The limitations of this study include the limited data points for PbtO2 as compared to similar studies in which PbtO2 is recorded by the minute. Additionally, our study would benefit from an increased patient population. Future studies may take into account these features to further delineate thresholds for PbtO2 treatment.Along with ICP monitors and monitoring in the assessment of CPP, brain tissue oxygenation allows yet another metric by which to optimize treatment in TBI patients. Previous methods of assessment of cerebral hypoxia relied on jugular bulb oximetry. Here, we describe the protocol used at our institution after the placement of brain tissue oxygen monitoring probes. We sought to keep PbtO2 levels above 28 mmHg for a good functional outcome. We demonstrate improvements in both GCS and GOS for patients who consistently had maximum PbtO2 levels above 28 mmHg on Days 3 to 5 while the monitoring system was in place. In addition, patients in this group had fewer deaths, in line with previous studies. However, this population did have a longer EVD duration as well as hospital stay duration. Future studies to better delineate thresholds should be performed.


The correct author name order is Xinzhi Wang1, Qianqian Jiang1,2, Yichi Zhang1, Nannan Yuan1 and Jianguo Tang1.The author name order was incorrectly displayed as Qianqian JiangThe authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.


Correction to: BMC Public Health (2020) 20:614https://doi.org/10.1186/s12889-020-08742-1It was highlighted that the original article  containeEthics approval and consent to participateThe QE team received initial approval for the AHME evaluation with “Exempt” status from UCSF’s Institutional Review on 13 June 2013. Ethical approval also was obtained from the Ghana Health Services Ethical Review Committee (ERC) and the Kenya Medical Research Institute (KEMRI). Prior to each round of data collection, the QE team received approval from all three ethical review boards for any changes made to the research protocol. Approvals for Round 2 (2017) of data collection were received: on 12 December 2017 from UCSF, 16 November 2017 from the ERC, and 17 January 2017 from KEMRI.Ethical approval for Round 1 of AHME’s Client Exit Interviews was obtained from Marie Stopes International’s Ethics Review Committee (MSI ERC) on 25 August 2017, the Ghana Health Service Ethics Review Committee (GHS ERC) on 20 November 2017, and Amref Health Africa Ethics and Scientific Review Committee (Amref ESRC) on 30 April 2018. Approvals for Round 2 were received from MSI ERC on 10 September 2018 and GHS ERC on 12 November 2018. The approval received from Amref ESRC on 30 April 2018 covered both Round 1 and Round 2 of data collection in Kenya.


GPR41 and GPR43 appear to be derived from the same image.After publication of this article , concernGPR41 adipose tissue. The original images for GPR41 and GPR43 adipose tissue are provided in The authors apologize that an error was made in generating An Academic Editor reviewed the updated figure and underlying data and confirmed that they support the results and conclusions reported in the published article.The authors apologize for the error in the published article.With this Correction, the authors also provide underlying data supporting Fig 2 and Fig 3 of , in S3 aS1 File(TIF)Click here for additional data file.S2 File(TIF)Click here for additional data file.S3 File(PZF)Click here for additional data file.S4 File(PZF)Click here for additional data file.


Sirtuin1 (Sirt1) has a NAD (+) binding domain and modulates the acetylation status of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC1α) and Fork Head Box O1 transcription factor (Foxo1) according to the nutritional status. Sirt1 is decreased in obese patients and increased in weight loss. Its decreased expression explains part of the pathomechanisms of the metabolic syndrome, diabetes mellitus type 2 (DT2), cardiovascular diseases and nonalcoholic liver disease. Sirt1 plays an important role in the differentiation of adipocytes and in insulin signaling regulated by Foxo1 and phosphatidylinositol 3′-kinase (PI3K) signaling. Its overexpression attenuates inflammation and macrophage infiltration induced by a high fat diet. Its decreased expression plays a prominent role in the heart, liver and brain of rat as manifestations of fetal programming produced by deficit in vitamin B12 and folate during pregnancy and lactation through imbalanced methylation/acetylation of PGC1α and altered expression and methylation of nuclear receptors. The decreased expression of Sirt1 produced by impaired cellular availability of vitamin B12 results from endoplasmic reticulum stress through subcellular mislocalization of ELAVL1/HuR protein that shuttles Sirt1 mRNA between the nucleus and cytoplasm. Preclinical and clinical studies of Sirt1 agonists have produced contrasted results in the treatment of the metabolic syndrome. A preclinical study has produced promising results in the treatment of inherited disorders of vitamin B12 metabolism. Sirtuin1 (Sirt1) is one of the seven mammalian proteins belonging to the silent information regulator 2 (Sir2) proteins/Sirtuin family with highly conserved catalytic and nicotinamide adenine dinucleotide (NAD+) binding domain . Sirt1 hCalorie restriction regulates the expression levels of Sirt1 in a tissue-dependent manner . Sirt1 rThe metabolic syndrome is a global public health problem related to overnutrition. It is defined as a cluster of cardio-metabolic abnormalities that includes obesity, insulin resistance or diabetes mellitus type 2 (DT2), hypertension and dyslipidemia ,22. The Secretion of insulin by pancreatic beta cells is enhanced by Sirt1 in response to glucose stimulation ,35,36. CClinical studies evidenced decreased Sirt1 expression in adipose tissues of obese patients ,42 and iSirt1 plays an important role in the differentiation of skeletal muscle . InsulinVisceral low grade inflammation triggered by adipocytes contributes to the pathogenesis of obesity, insulin resistance and other outcomes of the metabolic syndrome ,59,60. BConverging evidences support an antagonistic crosstalk between Sirt1 and nuclear transcription factor Kappa B (NF-κB) . DamaginMice with hepatocyte specific knock out of Sirt1 (Sirt1LKO) exposed to a high fat diet develop hepatic inflammation and ER stress . Liver iOxidative stress is associated with pathogenesis of complex metabolic diseases. Sirt1 activates Foxo transcription factors via the feedback loop ,76. ActiThe fetal programming hypothesis , also naThe decreased expression of Sirt1 plays a prominent role in the pathomechanisms of fetal programming produced by MDD in rats (vitamin B12 and folate) ,81,93,94MDD during pregnancy and lactation induces impaired fatty acid oxidation, reduced activity of complexes I and II, cardiac hypertrophy with enlargement of cardiomyocyte and liver steatosis in weaning rat pups ,96. ThesThe impaired cellular availability of vitamin B12 leads to ER stress related to decreased expression of Sirt1. ER stress is evidenced by increased expression and activation of elf2-α and activating transcription factor 6 (ATF6) and may be favored by decreased expression of heat shock proteins (HSP). Decreased Sirt1 impairs the transcription of HSP by increasing the acetylation of heat shock protein factor 1 HSF1;  93]. Co. Co93]. Preclinical and clinical studies in the treatment of complex and inherited metabolic diseases have produced contrasted results in the evaluation of health benefits of activation of Sirt1 by natural and pharmaceutical small activating molecules.Resveratrol is a natural polyphenolic compound found in red wine and grape and in plants and fruits like berries and peanuts. Resveratrol activates Sirt1 and mimics the caloric restriction status known to be protective against the metabolic syndrome. For decades, the therapeutic use of resveratrol has been considered in regard to its anti-inflammatory, antioxidant, antiaging and anticancer properties ,102,103.Given the health benefits of resveratrol in experimental animal studies, clinical trials have been carried out to evaluate its effects in the prevention and treatment of metabolic syndrome. A preliminary exploratory trial showed that administration of 500 mg resveratrol twice per day for 60 days in type 1 diabetic patients decreased fasting plasma sugar (FPS) and hemoglobin A1C . A prospfa/fa rats [ob/ob mice by upregulating hepatic Sirt1 and GCN5 [Activators of Sirt1 such as SRT1720, SRT2183 and SRT1460 are 1000-fold more active than resveratrol. SRT1720 and resveratrol improve insulin sensitivity in nutritionally and genetically induced obesity and DT2 in mice . SRT1720/fa rats . SRT1720/fa rats . Further/fa rats . SRT1720/fa rats . SRT1720and GCN5 . A compuand GCN5 . At low and GCN5 . Recentland GCN5 .Phase 1 clinical trials showed that SRT2104, another small molecule activator of Sirt1 is safe and tolerable in healthy volunteers, including the elderly ,133. HowMTR-KO mice with brain specific invalidation of Mtr gene encoding MS enzyme [Some severe forms of inherited disorders of intracellular metabolism of vitamin B12 are resistant to conventional treatments. Decreased Sirt1 activity plays a central role in some of the pathomechanisms of these disorders. We therefore evaluated the effect of Sirt1 agonists in a preclinical study in fibroblasts from patients with cblG and cblC inherited defects of vitamin B12 metabolism and an original transgenic mouse model of methionine synthase deficiency specific to neuronal cells. Patient fibroblasts with cblC and cblG defects of vitamin B12 metabolism presented with endoplasmic reticulum stress, altered subcellular localization of HuR, HnRNPA1 and RBM10, global mRNA mislocalization and increased HnRNPA1-dependent skipping of interferon regulatory factor 3 (IRF3) exons. SRT1720 inhibited ER stress and rescued RBP and mRNA mislocalization and IRF3 splicing. Furthermore, Sirt1 activation by SRT1720 partially restored the methylation and phosphorylation of these RBPs in patients’ fibroblasts. Interestingly, SRT1720, vitamin B12 and SAM treatment improved cognitive functions in conditional S enzyme . In partIn summary, preclinical and clinical studies of Sirt1 agonists have produced contrasted results in the treatment of the metabolic syndrome. A preclinical study has produced promising results in the treatment of inherited disorders of vitamin B12 metabolism.Numerous experimental evidence show a strong link between decreased Sirt1 and the pathological manifestations of metabolic syndrome by synergistic cellular and molecular mechanisms. It is noteworthy that over nutrition and MDD both produce a decrease of Sirt1. There are additive and synergistic effects of the MDD fetal programming and subsequent exposure to a high fat diet in adult life. The therapeutic prospects for using activators of Sirt1 in the treatment of disease outcomes of metabolic syndrome have not been conclusive to date. However, pharmacological activation of Sirt1 opens promising perspectives for the treatment of rare diseases of vitamin B12 metabolism with particular effects on reticulum stress and mislocalization of RBPs.