HESB). HES proteins are transcription factors that are key mediators of the Notch signaling pathway and gamma-secretase activity is crucial for the activation of this pathway. So, based on the RNA-Seq results we subsequently treated spinal cord injured larval sea lampreys with a novel gamma-secretase inhibitor (PF-3804014). This treatment also reduced the expression of HESB in the brainstem and significantly enhanced the regeneration of individually identifiable descending neurons after a complete spinal cord injury. Our results show that gamma-secretase could be a novel target to promote axon regeneration after nervous system injuries.In a recent study, we showed that GABA and baclofen (a GABAB receptor agonist) inhibit caspase activation and promote axon regeneration in descending neurons of the sea lamprey brainstem after a complete spinal cord injury . Now, we In mammals, including humans, spinal cord injury (SCI) is a devastating event that can lead to permanent disability. The death of neurons that are not replaced after the injury and lack of axon regeneration of axotomized neurons are two of the main causes of permanent neurological deficits after SCI. In striking contrast to mammals, lampreys, as other basal vertebrates, are able to recover locomotion spontaneously after a complete SCI . During In a recent study, we showed that endogenous GABA promotes axon regeneration in descending neurons of lampreys after a complete SCI, and that GABOB  and baclofen (a GABAB receptor agonist) treatments further promoted axon regeneration , 2019. GHES gene. HES proteins are key mediators of the Notch signaling pathway , and were performed in accordance with European Union and Spanish guidelines on animal care and experimentation. Animals were deeply anesthetized with 0.1% MS-222  in lamprey Ringer solution before all experimental procedures.All experiments involving animals were approved by the Bioethics Committee at the University of Santiago de Compostela and the Petromyzon marinus L. , were used in this study. Larval lampreys were collected from the river Ulla , with permission from the Xunta de Galicia, and maintained in aerated freshwater aquaria at 15°C with a bed of river sediment until their use in experimental procedures. Larval lampreys were randomly distributed between the different experimental groups.Mature and developmentally stable larval sea lampreys, SCI surgical procedures and drug treatments with GABA and baclofen were performed as previously described . Brieflyn = 16) and PF-3804014 (n = 21) treated animals using Neurobiotin  as a retrograde tracer as previously described  was used to acquire images of the whole-mounted brains/spinal cords. The identity of regenerated (Neurobiotin labeled) and non-regenerated identifiable descending neurons was determined for each brain. Giant descending neurons are always identifiable by their know location and big size  regardleFor axon quantification, we scanned the rostral spinal cord (starting at the obex) using a 20x objective. Then, we traced a horizontal line through the middle of the spinal cord confocal stack  using th® ND-1000 spectrophotometer . RNAs were stored at −80°C until use for RNA-Seq.After the GABA or baclofen treatment periods (29 days in the GABA experiment and 9 days in the baclofen experiment) the brainstems of control and treated larvae were dissected out and immediately put in RNAlater  . RNA extn = 24 in total) were sent to the Wellcome Trust Centre for Human Genetics  for library preparation and sequencing. RNA quality was checked again in a Bioanalyzer (Bonsai Technologies). All samples had RNA integrity number (RIN) of >8 . mRNA sequencing libraries were prepared from poly dT selected mRNA using the Illumina TrueSeq Stranded mRNA Library Preparation kit according to the manufacturer’s protocol. The average insert size of the libraries was approximately 300 bp. A different barcode was used for each sample to enable demultiplexing of the sequencing data. Sequencing was conducted on an Illumina HiSeq 4000 as 150 bp paired-end reads, yielding 23.2 to 27.8 million reads per sample.In the GABA experiment, 3 GABA treated and 3 control samples  were sent to Edinburgh Genomics  for library preparation and sequencing. RNA quality was checked again in a Bioanalyzer (Bonsai Technologies). All samples had RNA integrity number (RIN) of >7.5. mRNA sequencing libraries were prepared from poly dT selected mRNA using the Illumina TrueSeq Stranded mRNA Library Preparation kit according to the manufacturer’s protocol. The average insert size of the libraries was approximately 300 bp. A different barcode was used for each sample to enable demultiplexing of the sequencing data. Sequencing was conducted on an Illumina NovaSeq S1 flowcell as 100 bp paired-end reads, yielding 26.8 to 37.6 million reads per sample.In the baclofen experiment, 5 baclofen treated and 5 control samples  was applied, and transcripts with corrected Reactome , convertPRJNA561093 (GABA) and PRJNA561123 (Baclofen). The constructed transcriptome is available in the NCBI Transcriptome Shotgun Assembly (TSA) database under accession GHVE00000000. Read counts for each transcript were deposited in NCBI Gene Expression Omnibus (GEO) and are available under accession number GSE137860.Raw FASTQ files for all samples were uploaded to the NCBI Sequence Read Archive (SRA), and are publicly available under BioProject accession numbers n = 4 animals) from control 1 wpl vehicle treated animals and 4 brainstems  from 1 wpl PF-3804014 treated animals were processed for qPCR analyses of HESB (GenBank accession number: MN833804) expression. Specific primers for targeted genes were designed using Primer3: HESB-forward AAAGCAAAGACGGGACAGGA, HESB-reverse GATGTCCGCCTTCTCCAGTT, GAPDH-forward CAGATTCGTCCAGCTCCGTT, GAPDH-reverse CCATTGTGGAGCGGATGTCA. qPCR was performed on a Stratagene Mx3005P thermocycler (Agilent Technologies) using Brilliant III Ultra-Fast SYBR Green QPCR Master Mix in a final volume of 12.5 μL following the manufacturer’s protocol with 1 μL of cDNA per reaction. Primer concentration was 300 nM and each sample was run in duplicate. The cycling parameters were: 50°C for 2 min, 95°C for 10 min, followed by 40 cycles of amplification at 95°C for 15 s and 60°C for 1 min. After amplification, a dissociation step was performed raising the temperature from 65 to 95°C to create a melting curve and ensure the presence of a single amplification product. qPCR data were obtained by the MxPro software (Agilent Technologies) and quantification cycle values (Cq) calculated for each replicate and then averaged to obtain the final Cq value. HESB Cq values were normalized using the reference gene (GAPDH), and fold changes were calculated for each sample using the average of the controls as reference.4 brainstems . A Fisher’s exact test was used to determine significant differences between control and PF-3804014 treated animals .Statistical analysis was carried out using Prism 6 . Data were presented as mean ± SEM. Normality of the data was determined by D’Agostino–Pearson omnibus test and all data passed the normality test. A Student’s In previous work, we showed that both GABA and baclofen promote axon regeneration after a complete SCI in lampreys . Here, wHESB in another lamprey species Lampetra planeri  amongst differentially expressed genes after both treatments  as compared to vehicle treated controls  to activate the transcription of target genes like the HES family of transcription factors see . So, basHESB) and a significant enrichment of the Notch signaling pathway after GABA and baclofen treatments. The fact that this was detected in 2 independent RNA-Seq studies carried out in different sequencing platforms, using 2 different GABAergic drugs and at 2 different time points after injury provided strong confidence on the possible importance of this signaling pathway during regeneration. Gamma-secretase activity is crucial for the release of NICD to activate the transcription of target genes like the HES family of transcription factors  were previously detected over the course of recovery in an RNA-Seq study after SCI in lampreys : lampreys . Previoulampreys . KLF7 prlampreys , which cPRJNA561093 (GABA) and PRJNA561123 (Baclofen). The constructed transcriptome is available in the NCBI Transcriptome Shotgun Assembly (TSA) database under accession GHVE00000000. Read counts for each transcript were deposited in NCBI Gene Expression Omnibus (GEO) and are available under accession number GSE137860.Raw FASTQ files for all samples were uploaded to the NCBI Sequence Read Archive (SRA), and are publicly available under BioProject accession numbers The animal study was reviewed and approved by the Bioethics Committee of the University of Santiago de Compostela Santiago de Compostela, A Coruña, Spain.DS-C and DR-S performed the SCI surgeries, drug treatments and brainstem dissections. VP-C performed the RNA extractions. DR performed the bioinformatic analyses. LS, MR, and AB-I supervised the study. MR and AB-I conceived the study. DR and AB-I wrote the manuscript. All authors have approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Keywords Image-guided surgery; Immunohistochemistry; Molecular imaging; Myxofibrosarcoma; Soft tissue sarcoma; Tumor endothelial marker 1(TEM1), Vascular endothelial growth factor (VEGF).Myxofibrosarcoma(MFS) is the most common soft tissue sarcoma(STS) in elderly patients. Surgical resection remains the main treatment modality but tumor borders can be difficult to delineate with conventional clinical methods. Incomplete resections are a common problem and local recurrence remains a clinical issue. A technique that has shown great potential in improving surgical treatment of solid tumors is tumor targeted imaging and image-guided surgery with near-infrared fluorescence. To facilitate this technique, it is essential to identify a biomarker that is highly and homogenously expressed on tumor cells, while being absent on healthy non-malignant tissue. The purpose of this study was to identify suitable molecular targets for tumor-targeted imaging of myxofibrosarcoma. Ten potential molecular targets for tumor targeted imaging were investigated with immunohistochemical analysis in myxofibrosarcoma tissue (n = 34). Results were quantified according to the immunoreactive score(IRS). Moderate expression rates were found for uPAR, PDGFRa and EMA/MUC1. High expression rates of VEGF and TEM1 were seen. Strong expression was most common for TEM1 (88.2%). These results confirms that TEM1 is a suitable target for tumor-targeted imaging of myxofibrosarcoma.  Myxofibrosarcoma is a relatively common sarcoma in the elderly that mainly arises in the extremities3. Primary MFS most often presents as a subcutaneous painless, slow growing nodule, but completely infiltrative growth patterns along fascial planes without formation of an evident tumor mass have also been described4. The low-grade lesions have a relatively low metastatic potential but show a significant propensity for local recurrences8. These recurrences show higher-grade histology in up to 50% of all cases compared to the primary tumor3. Subsequently, high and intermediate grade disease exhibits a greater metastatic potential, mainly metastasizing to the lungs and lymph nodes9. Current guidelines recommend wide surgical resection combined with pre or post-operative radiotherapy as the preferred treatment10. Despite this strategy, positive margins after surgery occur in up to 20% of cases and reported local relapse rates range from 15 to 65%11.Myxofibrosarcoma (MFS) is a histological subtype of soft tissue sarcoma (STS) formerly classified as a myxoid-type malignant fibrous histiocytoma. It has been reclassified and defined as a distinct pathological entity in the World Health Organization (WHO) criteria set in 200213. These extensions are thought to correlate with microscopic spread of the tumor along fascial planes and have been found extending up to 3 centimeters away from macroscopically visible tumor borders15. Unfortunately, intraoperatieve delineation of these tumor borders and extensions based on tactile and visual clues is arduous and not always possible. Therefore, there is a need for accurate and real-time imaging techniques that can aid in intraoperative detection of disease.Currently clinicians rely on conventional imaging modalities such as CT and MRI to determine the location and extent of tumor burden prior to surgery. Translating these conventional imaging modalities to the surgical theatre remains challenging, forcing the surgeon to depend mainly on tactile and visual cues during the procedure. Previous imaging studies have shown that MFS has a tendency to grow in so-called ‘’tail sign” curvilinear extensions that arise from the main tumor16. To facilitate this technique it is essential to identify a biomarker that can be targeted with an imaging agent. This biomarker needs to be abundantly and homogeneously expressed on tumor cells while being absent on benign and healthy tissue. Another prerequisite is that the biomarker’s expression on tumor cells is not significantly altered by preoperative radiotherapy since this is often used for MFS. For a variety of tumor types there are established suitable targets for molecular imaging, but for MFS specific immunohistochemical expression profiles have not yet been described20.A promising technique that could aid in real-time tumor detection during surgery is tumor-targeted near-infrared (NIR) fluorescence imaging with targeted molecular agents29. The biomarker with the highest expression rate was subsequently evaluated on a selection of whole slides to evaluate the contrast between tumor tissue and adjacent benign regions. The secondary objective was to determine whether the expression of these biomarkers is influenced by preoperative treatment.Therefore, the primary objective of this study was to determine suitable biomarkers for targeted molecular imaging in myxofibrosarcoma using immunohistochemical analyses of surgically obtained tumor specimens. Ten biomarker candidates were selected based on the literature and availability of imaging agents that specifically target these biomarkersFormalin fixed paraffin embedded (FFPE) tissue samples of all patients diagnosed with MFS who underwent surgical resection in the Radboudumc between 2008 and 2015 were reviewed. Patient characteristics are depicted in Supplementary Table A total of 10 biomarkers was selected based on literature that reports on tumor targeted imaging of these markers, and the availability of a clinical grade targeting molecule or imaging conjugate . The following biomarkers were selected: Human epidermal growth factor receptor 2 (HER-2), carcino-embryonic-antigen (CEA), epithelial cell adhesion molecule (EpCAM), epidermal growth factor receptor (EGFR), epithelial membrane antigen , urokinase plasminogen activator receptor (uPAR), platelet-derived growth factor receptor α (PDGFRα), vascular endothelial growth factor A (VEGF-A), Tumor endothelial marker 1 (TEM1) and carbonic anhydrase IX (CA-IX).2O2 in 10Mm phosphate buffered saline (PBS) for 10 min at room temperature. Subsequently, tissue sections were washed with 10 mM PBS and stained with primary antibodies at room temperature or 4 °C respectively. Primary antibodies used and their characteristics are depicted in Supplementary Table A dedicated sarcoma pathologist (UF) at our tertiary referral center reviewed all histological slides and selected regions of interest for the tissue microarrays (TMAs). TMAs with 2 mm cores were sampled and constructed by a dedicated medical technician. Subsequently, TMAs were deparaffinized with xylene, rehydrated in ethanol and rinsed in distilled water according to standard local protocol. Heat-induced antigen retrieval was performed in 10 mM sodium citrate buffer (pH 6.0) or EDTA solution (pH 9.0) for 10 minutes in a microwave or in a PT Module. Endogenous peroxidase activity was blocked with 3% H30. The IRS is calculated as follows: Final expression score = percentage of positive cells multiplied by the staining intensity. This results in a final score ranging from 0–12. The final expression score was divided into 4 categories . Staining was scored on membranous expression, except for VEGF which was scored on cytoplasmic expression. Strong biomarker expression was considered as highly suitable for tumor-targeted imaging and image-guided surgery purposes. Evaluation of biomarker expression was performed independently by 2 trained observers, one dedicated sarcoma pathologist and one assistant professor of sarcoma research (U.F. and Y.V.). All cases where inter-observer disagreement occurred were discussed together with a third observer (J.M.G) until agreement was reached on the final expression score. Representative images of these expression intensity scores are depicted in Fig. The extent of biomarker expression was assessed in primary tumor tissue on a scale from 0–4. . For this study we combined the percentage and intensity scores into a final expression score according to the immunoreactive score(IRS) scoring methodAll FFPE blocks selected for the TMAs were evaluated to select those that represented a section of MFS with a clearly distinguishable border between malignant and benign/preexistent tissue. 4 µm sections were stained for H&E and the biomarker with the highest rate of expression found in the previous experiment. Tumor borders and invasive extensions were defined and marked by a dedicated sarcoma pathologist (U.F.) on the H&E slide. The immunostaining on tumor versus various types of benign tissue was assessed by the pathologist.Descriptive statistics were calculated and shown for expression of all biomarkers. Samples were also divided in 2 groups, those that were treated with or without neoadjuvant radiotherapy. Differences in biomarker expression between these groups were calculated and compared using the Mann-Whitney U test. Results were considered statistically significant when the p value was smaller than 0.05. All statistical analyses were performed using the SPSS  and GraphPad Prism 6 .This study was conducted according to the ethical standards laid down in the 1964 Declaration of Helsinki and in accordance with the ethical standards of the local ethics committee and with national Dutch guidelines . The local medical ethics committee at the Radboud university medical center, Nijmegen, the Netherlands, approved the study prior to its conduction. The need for informed consent was waived by the institutional review board.Cases with inconclusive pathology results were re-evaluated. In five cases the diagnosis MFS was not certain and these were excluded from further analysis. A total of 34 cases with proven diagnosis of MFS were included in the TMA and staining protocol. Ten of these 34 cases were treated with preoperative radiotherapy Per case a minimum of 2 and a maximum of 3 samples per FFPE block were taken and processed in the TMAs. All cases expressed TEM1, with strong expression being present in 30 cases (88.2%). For VEGF, 33 (93%) of all cases showed expression but only 7 (20.6%) exhibited strong expression. EMA/MUC-1 staining was seen in 22 (57.6%) cases with 7 (20.6%) exhibiting strong expression. Only 2(5.9%) of the 10 cases that stained positive for CAIX demonstrated strong expression. Expression of PDGFRa was observed in 26(76.5%) cases but strong expression occurred only once. 12(25.3%) cases stained positive for UPAR but strong expression was seen in 1(2.9%) case. EGFR expression was observed in 9 cases (26.5%) but no strong expression was seen. None of the cases demonstrated expression of EpCAM, CEA or HER2NEU. The expression rates and corresponding percentages of all tumor markers are depicted in Fig. Higher rates of strong expression after preoperative treatment with radiotherapy were seen for TEM1 but this was not statistically significant (p = 0.176). The expression pattern of EMA/MUC1 seems to be negatively influenced by preoperative treatment but this was also not statistically significant (p = 0.181). No significant effect of neoadjuvant radiotherapy on biomarker expression was observed in this cohort. Results of treatment effect are shown in Table Ten FFPE blocks showed clear tumor to benign tissue borders and were selected for further analysis. Tissue sections of 4 µm were stained for H&E and TEM1. All sections showed invasive MFS next to various benign/preexistent tissues such as subcutaneous fat, skeletal muscle and adjacent fascia, epidermis, peripheral nerve bundles, blood vessels and necrotic tumor regions. Clear contrast was seen between MFS and benign/preexistent tissue in all sections stained with TEM1. Muscle, fascia and subcutaneous fat showed no discernable TEM1 expression. Weak expression was seen in normal endothelium and fibrous tissue but strong contrast to tumor tissue was still present. Two sections revealed microscopic tumor involvement of the septa highlighted by strong TEM1 expression. Three representative examples are shown in Fig. In this immunohistochemical study the expression of 10 biomarker candidates in MFS was examined in surgical samples of 34 patients. Moderate expression rates were observed for uPAR, PDGFRa and EMA/MUC1 and high expression rates of VEGF and TEM1 were observed. Strong overexpression of TEM1 was observed in 89% of all cases, demonstrating great potential as a biomarker for targeted approaches for diagnosis and treatment.33. As a result of these trials, a variety of suitable biomarkers have been identified for NIR-fluorescence guided surgery. For soft tissue sarcoma, targeted NIR- fluorescence-guided surgery is still mostly uncharted territory and the literature on tumor marker expression is very limited, especially for MFS.Targeted NIR fluorescence imaging has already been studied in several clinical trials for colorectal, head and neck, pancreatic and renal cancer and has shown its potential and added value in intraoperative decision making34. Scoccianti et al.35 reported on EMA/MUC1 expression in MFS and found no positive staining for EMA/MUC-1. The difference of the results reported in this manuscript might be attributed to a variety of factors such as the use of different antigen retrieval techniques and the use of a different primary antibody. Forker et al.36 investigated a selection of hypoxia markers, including CAIX, in a large cohort of soft tissue sarcomas. The cohort also contained 52 myxofibrosarcomas but the CA-IX expression was only reported for all STS types together. Mentzel et al.37 studied vascularity and tumor progression in a series containing 43 MFS cases. They used mRNA in situ hybridization (ISH) to study VEGF expression and report that all cases showed some extent of VEGF expression. This is in accordance with our results, but regrettably, VEGF expression was only studied in 7 of their 43 cases. Sato et al.38 studied the expression of EGFR and ERB2(HER2NEU) in Japanese myxofibrosarcoma cases. They did not find HER2NEU expression but report that 98% of cases shows EGFR expression. This is a surprising lack of compatibility with the EGFR expression rates in our cohort, where EGFR expression was only found in 26.5%. Because of this discrepancy, an additional EGFR staining with a different primary antibody was performed in our cohort but again no EGFR expression was found. The difference might be explained by genetic differences, the use of a different primary antibody or variability in antigen retrieval techniques used.Small studies performing expanded molecular profiling of MFS are described in the literature but immunohistochemical studies that specifically investigate potential targets for tumor-targeted imaging are lackinget al.39. This TASC scoring system corrects for a variety of factors, such as extracellular localization of the biomarker, internalization of the target, previous use of the biomarker in in vivo imaging studies and enzymatic activity in or around tumor tissue. The final maximum TASC score is 22 points and a score ≥18 is considered suitable for imaging purposes. When applying the TASC criteria to TEM1, the final score of 21 confirms its high suitability as a target for NIR fluorescence-guided surgery. The second best marker in our cohort (VEGF) is an interesting and applicable target for NIR-Fluorescence image-guided surgery since a fluorescently-labeled tracer targeting VEGF (bevacuzimab-IRDye800CW) is clinically available and has been extensively studied for several malignancies40. The main drawback of VEGF is that it is a signal protein produced to stimulate the formation of blood vessels. Therefore the protein may also be found in benign tissue, especially in injured regions with high rates of angiogenesis41. Because of some of these characteristics, the final TASC score of VEGF is 17, which is just below the threshold of 18 set by Oosten et al.39.The suitability of a biomarker for MFS is not solely dependent on its upregulation but influenced by several factors. A scoring system to objectify the assessment of biomarker suitability for targeted NIR-Fluorescence image-guided surgery has been proposed by Oosten 42. The fact that muscular fascia did not show TEM1 expression is also of interest because it suggests that TEM1 targeted fluorescence imaging can allow the surgeon to assess if there is tumor involvement of the fascia. The surgeon can follow the muscular fascia as a surgical plane to guide the resection when tumor involvement is not detected. Peripheral nerve bundles were also negative for TEM1, allowing for identification and possible sparing of vital nerves during procedures and therefore reduce surgical morbidity.The absence of the biomarker in benign tissue mentioned in these TASC criteria remains one of the most pivotal characteristics of a suitable biomarker. TEM1 immunohistochemistry on complete tissue sections shows that expression was absent or very limited on a variety of benign/preexistent tissues such as subcutaneous fat, epidermis, muscle and fascia while the primary tumors with diffuse infiltration along preexisting fibrous septa was strongly positive for TEM1. This is of great clinical importance since MFS very frequently extends along the septa into resection margins, with one third of all MFS tumors being larger than originally expected during surgical resection44. While TEM1 is known to be expressed on tumor cells of several sarcoma subtypes44, the expression patterns in other cancer types are generally of a different nature. In melanoma, ovarian, breast, lung and brain cancers TEM1 is mainly found to be highly expressed on perivascular and stroma cells48. This does not mean that targeting TEM1 is not feasible for these cancer types because stromal targeting also yields potential for image-guided surgery19. Besides intraoperative imaging, TEM1 is also an attractive target for preoperative imaging. A preclinical trial investigating TEM1-targeted 89Zr-Immuno-PET with the clinical grade monoclonal antibody ontuxizumab has shown the possibility to determine TEM1 status in a murine model49. This could potentially facilitate non-invasive selection of patients suitable for TEM1 targeted NIR- Fluorescence image-guided surgery, systemic therapy with antibodies or antibody-drug-conjugates44. TEM1 targeted immuno-PET may also have potential as a diagnostic tool for metastases detection of STS, since these retain TEM1 expression50.A potential advantage of a fluorescent agent targeting TEM1 is that it may be used for a variety of oncological indications. The development of clinical grade tracers for tumor-targeted NIR-fluorescence imaging is an expensive and lengthy process so ideally the developed agent targets a biomarker that is (over)expressed on multiple tumor types. TEM1 is a target with such characteristics since it is strongly expressed on tumor cells, tumor vasculature and stroma in the majority of soft tissue and bone sarcomas51.The present study contains several limitations. First, the analyzed cohort is relatively small because MFS is a rare disease. Therefore the analysis of preoperative treatment effect on expression patterns lacks sufficient statistical power. Nevertheless, to our knowledge this study is the first cohort of patients with MFS analyzed for biomarkers for tumor-targeted imaging. Second, the selection of biomarkers was not completely comprehensive, also because selection was partially based on availability of clinical grade targeting agents or imaging conjugates. Finally, because TMAs were used for staining purposes, adjacent benign tissue such as muscle, fat or fascia was not investigated for all markers. However, the marker with the highest expression (TEM1) was extensively investigated for expression in a variety of benign adjacent tissues, emphasizing its potential as a biomarker for image-guided surgery. These results are supported by the literature reporting on the absence of TEM1 expression in benign and healthy tissueTumor -targeted image-guided surgery has the potential to improve surgical treatment of myxofibrosarcoma. In this immunohistochemical study we investigated a variety of biomarker candidates to determine a suitable target for NIR fluorescence image-guided surgery of myxofibrosarcoma. The results clearly show that TEM1 is an excellent potential biomarker to investigate in future studies.Supplementary Dataset 1.


To that end cortisol levels in different tissues of fed and fasted adults were measured using ELISA, open chromatin in adult blood cells was mapped using ATAC-seq, and gene activity in adult blood and brain cells was measured using qRT-PCR.Chronic early life stress can affect development of the neuroendocrine stress system, leading to its persistent dysregulation and consequently increased disease risk in adulthood. One contributing factor is thought to be epigenetic programming in response to chronic cortisol exposure during early development. We have previously shown that zebrafish embryos treated chronically with cortisol develop into adults with constitutively elevated whole-body cortisol and aberrant immune gene expression. Here we further characterize that phenotype by assessing persistent effects of the treatment on cortisol tissue distribution and dynamics, chromatin accessibility, and activities of glucocorticoid-responsive regulatory genes klf9 and fkbp5 in blood and brain.Adults derived from cortisol-treated embryos have elevated whole-body cortisol with aberrantly regulated tissue distribution and dynamics that correlate with differential activity of  Epidemiological studies have shown that early life stress (ELS) can increase later life risk of developing a variety of physical and mental health problems, many of which are linked to chronic inflammation –3. This Zebrafish are emerging as an excellent model system for experimental studies of stress-induced developmental programming –18. We rZebrafish embryos were treated with cortisol or vehicle as described previously , then rapomca, a target of GR-mediated repression, was significantly lower in fasted fish derived from cortisol-treated embryos and is less affected by Dex  were found in our previous study to be highly upregulated in larvae chronically exposed to cortisol  of both genes was carried out on blood cells of adults derived from cortisol-treated embryos and their vehicle-treated siblings. In 14 out of 17 independent blood cell samples, klf9 and/or fkbp5 expression was elevated in the cortisol-treated fish, both genes being overexpressed ~ twofold on average  may have been recently stressed. Chromatin immunoprecipitation detected higher levels of H3K4 trimethylation in the promoter regions of both genes in blood cells of the treated fish . These results underscore the relevance of zebrafish as a model organism for investigating the mechanisms underlying developmental programming and neuroendocrine dysregulation associated with ELS and chronic glucocorticoid exposure in humans.These results extend our earlier findings showing that in zebrafish, chronic glucocorticoid exposure during early development leads to persistent dysregulation of the neuroendocrine stress system and associated gene expression in adulthood. The data indicate that the neuroendocrine dysregulation occurs a multiple levels, including cortisol production (higher in treated fish), delivery of cortisol from circulation to tissues , and activity of key genes that regulate GC signaling dynamics in receiving cells . Cells were cultured at 28° C in 5% CO2. Blood cell viability was assessed by cell count with a hemocytometer. Zebrafish blood cells were determined to be stable in culture for at least 5 days.Blood was collected by a nonlethal venous puncture under the operculum. 1µL samples of blood from 6 fish of mixed sex per condition were pooled in heparin (5 I.U./mL) in PBS, washed, and plated at a density of 1 million cells/mL in supplemented L-15 media  from three biological replicates of adults derived from cortisol-treated embryos and their control (vehicle-treated) siblings  were constructed and sequenced and the sequence reads processed as described in the Additional file Total RNA was isolated from blood cells or dissected brain tissue using Trizol and ethanol precipitation. qRT-PCR was performed and the data quantified by the ddCt method as previously described .klf9  and fkbp5 . A standard curve was made using serial dilutions of control input DNA and regression analysis was used to calculate percent recovery (immunoprecipitated DNA/input DNA) × 100%.ChIP was performed on formaldehyde-crosslinked chromatin from blood cells, essentially using the procedure described by Lindeman et al.  an anti-t test.Cohort sizes were determined by power analysis in R with the “pwr” package, using a Cohen’s d effect size calculated from previous experiments of cortisol measurements in adult zebrafish treated as larvae with 1 uM cortisol. This analysis indicated that a minimum n of 5 is needed to detect statistically significant (p < 0.05) differences between treatment and controls. Some experiments had multiple biological replicates, whereas others were done only once with technical replicates, as noted in the Figure legends. Tissue cortisol measurements in Fig. The measurements shown in Fig. When interpreting the single time point measurements reported here it is important to consider that cortisol levels are dynamic, with circadian rhythms as well as ultradian fluctuations with an order of magnitude dynamic range.Additional file 1: ANOVA of tissue cortisol levels shown in Fig. Additional file 2: Figure S1. Top ten transcription factor binding motifs identified by HOMER motif enrichment analysis of sequences from the 251 peaks that scored ≥ 100.Additional file 3: Figure S2. Survey of gene expression by 96-well qPCR array and relative expression levels of klf9 and fkbp5 in brain tissue dissected from different experimental cohorts. Each measurement was made from pooled brain RNA of 6 individuals of mixed sex , except where indicated (panel f). The qPCR measurement shown in panel (b) is from the same RNA samples that were used to generate the data depicted in panel (a).Additional file 4: Methods. ATAC-seq library construction protocol, sequencing and read processing.


Rationale: Liver fibrosis is frequently associated with gut barrier dysfunction, and the lipopolysaccharides (LPS) -TLR4 pathway is common to the development of both. Intestinal alkaline phosphatase (IAP) has the ability to detoxify LPS, as well as maintain intestinal tight junction proteins and gut barrier integrity. Therefore, we hypothesized that IAP may function as a novel therapy to prevent liver fibrosis.Methods: Stool IAP activity from cirrhotic patients were determined. Common bile duct ligation (CBDL) and Carbon Tetrachloride-4 (CCl4)-induced liver fibrosis models were used in WT, IAP knockout (KO), and TLR4 KO mice supplemented with or without exogenous IAP in their drinking water. The gut barrier function and liver fibrosis markers were tested.Results: Human stool IAP activity was decreased in the setting of liver cirrhosis. In mice, IAP activity and genes expression decreased after CBDL and CCl4 exposure. Intestinal tight junction related genes and gut barrier function were impaired in both models of liver fibrosis. Oral IAP supplementation attenuated the decrease in small intestine tight junction protein gene expression and gut barrier function. Liver fibrosis markers were significantly higher in IAP KO compared to WT mice in both models, while oral IAP rescued liver fibrosis in both WT and IAP KO mice. In contrast, IAP supplementation did not attenuate fibrosis in TLR4 KO mice in either model.Conclusions: Endogenous IAP is decreased during liver fibrosis, perhaps contributing to the gut barrier dysfunction and worsening fibrosis. Oral IAP protects the gut barrier and further prevents the development of liver fibrosis via a TLR4-mediated mechanism. Fibrosis is a common sequela in many chronic liver diseases, including hepatitis, benign or malignant bile duct obstruction or alcoholic liver disease. In recent years, an important relationship between liver pathology and the gastrointestinal tract has come to light Intestinal alkaline phosphatase (IAP) is one subtype of the alkaline phosphatase family and is produced exclusively in the intestine with highest expression in the duodenum IAP is known to detoxify bacterial LPS by removing a phosphate group from its lipid A moiety, thus preventing recognition by its receptor TLR4 Stool samples were collected from patients with liver cirrhosis hospitalized in the Liver Disease Center at the Second Affiliated Hospital of Xi'an Jiaotong University. Control stool samples were collected from patients diagnosed with inguinal hernia, gallstones, and thyroid nodular goiter and with no cirrhosis seen in the surgical clinic at Department of General Surgery, Second Affiliated Hospital of the Xi'an Jiaotong University before their operation.IAP KO mice (C57BL/6 background) mice were generated by the Sanford-Burnham Medical Research Institute  IAP was dissolved in autoclaved tap water to a final concentration of 200 IU/mL. IAP was given 4 days prior to bile common duct ligation or CCl4 injection and then continued for the remainder of the experiment. The drinking water supplemented with IAP was changed every day.2; 10 μM ZnCl2) then homogenized with glass beads for 10 min. The homogenate was then centrifuged at 10,000 g for 10 min and the supernatant was taken for analysis. Total protein concentration was measured by the Bradford assay. To measure IAP activity, 25 μL or supernatant was added to 175 μL pNpp solution  and OD405 was measured over time. In separate wells, the selective IAP inhibitor phenylalanine (10 mM) was added to the pNpp solution. Bovine IAP (Sigma) was used to create a standard curve. Change in OD405 over time was normalized to total protein concentration for each sample, and the result with phenylalanine was subtracted from the result without phenylalanine. The final result was reported as units IAP/mg protein.Stool samples were diluted at a ratio of 1:30 (weight:volume) by stool dilution buffer  was used to analyze the data, shown as mean ± SD, and a two-tailed student's Other detailed methods can be found in the Table A total of 18 control patients without cirrhosis and 86 patients with liver cirrhosis had stool samples collected. The mean age of liver cirrhosis patients was 48.08 ± 10.16 with 51 males and 35 females. The etiology of liver diseases included hepatitis B virus (n= 53), hepatitis C virus (n= 12), HBV and HCV (n=3), alcoholic cirrhosis (n=6), autoimmune cirrhosis (n=5), cholestatic cirrhosis (n=4), and unknown causes (n=3). The Child-Pugh classifications of these patients were as follows: 27 class A patients, 30 class B patients, and 29 class C patients. The non-cirrhosis control samples were collected from patients with biliary colic (n=8), inguinal hernia (n=6) and thyroid nodular goiter (n=4) with a mean age of 45.39 ± 12.52. The liver function was class A in all control group patients. The characteristics of these patients are shown in p<0.05, Figure p<0.05, Figure Compared to patients without cirrhosis, patients with cirrhosis had significantly lower fecal IAP activity (p<0.05), and activity remained reduced when measured at both 2 weeks and 3 weeks after surgery   Figure D. Compar) Figure E-F.Impairment of the gut barrier is a critical component in the development of liver fibrosis. IAP has been shown to play a critical role in maintaining intestinal tight junction protein expression. We therefore tested the gut barrier function in CBDL and CCl4 injected WT and IAP KO mice. Terminal ileum mRNA expression of ZO family and Occludin were significantly decreased in WT and IAP KO mice after both CBDL and CCl4 injection. At 3 weeks post-CBDL, ZO-2, ZO-3 and Occludin decreased more in WT mice than in IAP KO mice    Figure A. Portal) Figure C. CCl4-t) Figure E-F.As the gut lumen is a rich source of bacterial toxins, especially LPS, and these gut-derived mediators have been shown to participate in the pathogenesis of chronic liver inflammation/fibrosis We next tested whether supplementation with IAP in the drinking water could help to maintain the gut barrier function in murine models of liver fibrosis. As shown in Figure As translocation of gut derived LPS is a key driver of liver fibrosis, we hypothesized that oral IAP would maintain the gut barrier, detoxify luminal LPS, and attenuate the liver fibrosis induced by CBDL and CCl4 injury. After 3 weeks and 8 weeks of IAP supplementation in the CBDL and CCl4-treated mice respectively, our data suggested that oral IAP significantly decreased expression of TIMP-1, Collagen-1 and ACTA-2 in both WT and KO mice Figure A-C, 6F-GLPS induces liver fibrosis by translocating across the gut barrier, into the portal vein, where it then enters the liver, and activates the TLR4 pathway resulting in a variety of downstream signaling that lead to fibrosis While it is known that LPS is a substrate for IAP, and that IAP decreases luminal LPS CCl4 and CBDL are two of the most commonly studied liver fibrosis animal models. These models begin with an insult to the liver, by either direct damage to hepatocytes (CCl4) or intrahepatic cholestasis (CBDL), followed by induction of key inflammatory pathways. Ultimately, this leads to the secretion of inflammatory cytokines, which in turn activate hepatic stellate cells via intrahepatic pathways thereby driving the development of fibrosis IAP is well-known as a marker of enterocyte maturation and its activity has been negatively correlated with intestinal inflammation While gut barrier function relies on many different factors, the central component of the intestinal barrier are enterocytes that are tightly bound to adjacent cells by apical junctional proteins that include claudins, occludins, E-cadherins, desmosomes and junctional adhesion molecules The key function of IAP is its ability to detoxify a variety of pro-inflammatory mediators that exist within the gut lumen, including LPS which is composed of hydrophilic polysaccharides within its core and O-antigen and a hydrophobic lipid A component. In recent years, LPS has been shown to be one of the key mediators linking the gut to the development of liver disease, as well as many other systematic diseases It has been shown that targeting gram negative bacteria and LPS by long-term administration of rifaximin, polymyxin or norfloxacin may rescue liver fibrosis Although IAP functions both to directly maintain the gut barrier and also to detoxify bacterial ligands such as LPS, it is not clear which function of IAP plays a more important role in its ability to attenuate liver fibrosis. We sought to address the mechanistic question by using the TLR4 KO mice. Our finding shows that the TLR4 KO mice did not have the same degree of improvement as wild type mice, suggests that this pathway is a critical one in regard to IAP action. With increased exposure of TLR4 to LPS and/or increased expression or sensitivity of TLR4, there is an inappropriate immune response which induces a chronic inflammatory liver disease In conclusion, our data show that IAP level decreased in liver fibrosis, likely contributing to a breakdown of the intestinal barrier. The gut barrier dysfunction results in an increased LPS translocation into the liver through the portal system, worsens liver damage, and creates a vicious cycle, and ultimately leads to liver fibrosis. We have shown that oral IAP could represent a novel therapy to prevent the development of liver fibrosis by detoxifying luminal LPS and protecting the gut barrier function, in a TLR4-dependent fashion.Supplementary material.Click here for additional data file.


In this comprehensive review we critically summarize the literature regarding the intriguing roles of NO, H2S and CO in the neuromolecular mechanisms of anxiety in the context of their putative, yet promising therapeutic application. A possible mechanism of gasotransmitter action at the level of anxiety-related synaptic transmission is also presented. Brain gasesous neuromediators urgently require further wide ranging studies to clarify their potential value for the current neuropharmacology of anxiety disorders.Although anxiety is perhaps one of the most significant current medical and social problems, the neurochemical mechanistic background of this common condition remains to be fully understood. Multifunctional regulatory gasotransmitters are novel, atypical inorganic factors of the brain that are involved in the mechanisms of anxiety responses. Nitric oxide (NO) signaling shows ambiguous action in animal models of anxiety, while NO donors exert anxiogenic or anxiolytic effect depending on their chemical structure, dose, treatment schedule and gas release rapidity. The majority of NO synthase inhibitors act as a relatively potent axiolytic agents, while hydrogen sulfide (H A receptor has been associated with anxiolytic activity, whereas its inhibition may trigger anxiety responses. The expression of several enzymes responsible for neural steroidogenesis, such as 5α-reductase, 3α-hydroxysteroid dehydrogenase and aromatase (3α-HSD), have been detected in the limbic structures  caused anxiogenic effects in rats in the EPM test  dimer (CORM-2) molecule promoted an anxiolytic-like effect (examined with EPM and LD tests) and increased HO-1 activity in the rat LC [One of the few pioneer studies on CO and anxiety was conducted by Carvalho-Costa et al. . This ime rat LC . Additioe rat LC .l-NAME, Kim and Rivier [By applying intracerebroventricular injections of tin protoporphyrin  and d Rivier  were abld Rivier . The samd Rivier .2) synthesized during intracerebral amino acid metabolism has also been very recently suggested as a new gaseous transmitter [2 a product of aspartate aminotransferase (AAT) catalytic activity can easily pass through all neurolemmal barriers and is considered to be involved in some cardiovascular regulatory processes including blood pressure control [2 significantly reduced neuronal injury in the rat hippocampus after chronic cerebral hypoperfusion (CCH) and improved cognitive disturbances in the Morris water maze. Of note, hippocampal catalase activity was also elevated suggesting that SO2 may support brain antioxidant responses [2 donors sodium sulfite and hydrosulfite (Na2SO3:NaHSO3) caused a distinct anxiolytic-like effect in mice (in the OFT test) under normal conditions and abolished chronic mild stress (CMS)-induced anxiety-like behavior. This may open a cautious discussion about SO2 as a new possible agent for the treatment of anxiety [Interestingly sulfur dioxide (SOnsmitter . Brain-d control . This noesponses . It was  anxiety , 138.2S and CO donors as anxioltytic factors using a large spectrum of behavioral methods. Importantly, a relatively narrow therapeutic window of these substances as well as their potential biphasic actions should also be taken into account. To conclude, an alternative way to treat anxiety has potentially been identified and it may introduce a new therapeutic strategy in current neuropsychiatry. Nevertheless, there are still numerous questions related to the anxiolytic properties of gaseous neurotransmitters and more advanced pharmacological studies are needed to confirm their possible clinical applicability.A number of reports have clearly demostrated that nitric oxide molecules are involved in the neurochemistry of anxiety responses in animal models and both NO donors and selective NOS inhibitors can be rightly considered in the future treatment of anxiety disorders. Moreover, recent findings have revealed that hydrogen sulfide and carbon monoxide may also exert distinct anxiolytic effects and its donors seem to be applicable. It should not be excluded that a novel way to treat anxiety has been postulated and may be an innovative alternative in the development of new therapeutic possibilities. However, there are still numerous concerns related to the pharmacological effects of gaseous neurotransmiter donors or modulators and further basic investigations are urgently required to prove their potential usefulness for the pharmacotherapy of anxiety. Firstly, all potential treatment schedules either acute or long-term should be precisely evaluated. Secondly, more detailed animal studies should assess the efficacy and safety-profile of NO, H


Arabidopsis pollen transcriptome analysis revealed new intergenic transcripts of unknown function, many of which are long non-coding RNAs, that may function in pollen-specific processes, including the heat stress response.Arabidopsis thaliana pollen exposed to heat stress (HS), we identified 66 novel and 246 recently annotated intergenic expressed loci (XLOCs) of unknown function, with the majority encoding lncRNAs. Comparison with HS in cauline leaves and other RNAseq experiments indicated that 74% of the 312 XLOCs are pollen-specific, and at least 42% are HS-responsive. Phylogenetic analysis revealed that 96% of the genes evolved recently in Brassicaceae. We found that 50 genes are putative targets of microRNAs and that 30% of the XLOCs contain small open reading frames (ORFs) with homology to protein sequences. Finally, RNAseq of ribosome-protected RNA fragments together with predictions of periodic footprint of the ribosome P-sites indicated that 23 of these ORFs are likely to be translated. Our findings indicate that many of the 312 unknown genes might be functional and play a significant role in pollen biology, including the HS response.The male gametophyte is the most heat sensitive of all plant tissues. In recent years, long noncoding RNAs (lncRNAs) have emerged as important components of cellular regulatory networks involved in most biological processes, including response to stress. While examining RNAseq datasets of developing and germinating The online version of this article (10.1007/s00497-020-00400-1) contains supplementary material, which is available to authorized users. High temperatures have profound harmful effects on plants’ reproduction, often causing severe damages to complete loss of crops   that can be translated into small polypeptides (> 100 amino acids), also known as micropeptides or microproteins, with growing evidence supporting that they are biologically functional, regulating target genes in Arabidopsis thaliana reference genome annotation, Araport11v4, significantly expanded the number of genes compared with the earlier TAIR10 annotation, including a massive increase in the number of long noncoding RNAs (lincRNAs and NATs) from 259 to 3559 genes. The updated annotation version also included 508 intergenic novel transcribed regions  from three independent RNAseq experiments with Arabidopsis thaliana (Col-0) were grown in 16:8 light regime and at 21 °C for 5 weeks until flowering had established. Pollen was harvested from open flowers using a customized pollen vacuum wand  according to the manufacturer’s recommendations. RNA was shipped to Beijing Genomics Institute  for multiplexed Illumina HiSeq 2000 paired-end sequencing. Sequencing was performed at 2 × 50 bp for all samples.A. thaliana reference genome using the spliced aligner tool Tophat2 (V.2.0.13) and Bowtie 2 (v.2.2.4).Sequence pairs were trimmed and filtered using Trimmomatic v. 032  were identified and mapped using the in-house services of the Nevada INBRE Bioinformatics Core at the University of Reno (USA). Briefly, genome alignments generated by Tophat2 were processed using the Cufflinks package (v.2.2.1) reference annotation-based transcript (RABT) assembly method. Predictions from the independent Cufflinks runs were combined using the cuffmerge command to produce a single set of predicted transcripts. To determine which of the unannotated transcripts were annotated in the Araport11 genome annotation, we used IntersectBed from the Bedtools suite  was performed using DESeq2 with the variance-stabilized normalized RNA-Seq data to validate the clear separation between the different conditions.Transcript counts were filtered to exclude those with < 10 counts in all samples. Filtered count data were then normalized via the median ratio method . First-strand cDNA synthesis was performed using qScript Flex cDNA synthesis kit (Quantabio). qRT-PCR was performed in CFX96Connect (Bio-Rad). The gene HTR5 (AT4G40040) served as a reference gene. Relative normalized expressed was calculated using 2−E value of <  = 1e − 05. We ran the search twice using the nr/nt database once excluding hits within Brassicaceae and second time only searching within Brassicaceae but eliminating hits to Arabidopsis thaliana.To understand the conservation of these novel transcripts, we performed megablast at the NCBI using the novel transcripts as the query and requiring an We then extracted the full taxonomic lineage for each hit and were then able to assign hits to a given level in the phylogenetic tree.http://cantata.amu.edu.pl/) was used to identify conserved lncRNAs   mature pollen (MP) and cauline leaves grown under control (22 °C) or exposed to heat stress cycle for 3 days, referred to herein as MP_HS dataset and CL_dataset, respectively. The analysis of the RNAseq filtered raw data was performed using an in-house de novo assembly pipeline with the TAIR10 genome build as reference  . Paired-end RNAseq data were generated from polyadenylated RNA from nce Fig.  section.nce Fig. , referrence Fig. , referre control 2 °C or eBased on the criteria of RNA longer than 200 nucleotides or having ORF(s) shorter than 100 amino acids , defined as being located 500 or more bp away from annotated protein-coding genes, not encoding transposable elements (TEs), and not overlapping with natural antisense transcripts , abscisic acid (ABA), methyl jasmonate (MeJA), or the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC). We, therefore, compared the coordinates of the newly identified TUs encoding genes in our pollen datasets with those of the genes identified in the Deforges et al.  to 1 RPM.Therefore, we set the threshold indicating We verified the expression of TUs in the RNAseq datasets using endpoint PCRs for five selected stress-responsive PEX with cDNA pooled from inflorescences grown under control and heat stress Fig. b.Among pollen-expressed genes, 269 and 213 from MP_HS and MP_Hot/Cold datasets, respectively, were shown higher than 10 mean RPM. All of these genes had none or low expression level (RPM < 1) in cauline leaves Table  RNAseq dA. lyrata and an additional four genes in three species of other Brassicaceae family members (Table S7). All the identified conserved genes were expressed in both MP libraries, and six were also expressed in germinating pollen. In addition, around half (18/34) were pollen-specific. However, no putative lncRNA homologs were identified from more evolutionarily diverse species, suggesting that the majority of TUs encoding loci identified here originated within the Brassicaceae lineage (Table S7). The relatively low number of lncRNA homologs hits from CANTATAdb 2.0 may result from the libraries used to construct the database, as it is curated from transcriptomic studies not including pure pollen or pollen-enriched samples.To search for conserved genes among the 312 XLOCs, we used CANTATAdb 2.0, a database of putative lncRNAs predicted from hundreds of RNAseq libraries from 39 species covering a broad diversity of land plant species . Among the nine phylogenetically oldest PEXs, belonging to Eudicotyledons and Magnoliopsida, six are pollen-specific.Because of the low number of matches obtained from CANTATA, we conducted a more comprehensive search for homologous genes using BLASTn megablast within the standard databases nucleotide collection (nr/nt). We then used the BLASTn results in a phylostratigraphic approach to determine the phylogenetic origin of each XLOC, assigning each XLOC to a phylostratum according to the oldest phylogenetic node to which the XLOC can be traced Fig. . We founThe induction of HSPs and HSFs in cauline leaves was severalfold higher than in the three pollen datasets, and the abundance (RPM) of the transcripts was 1–2 orders of magnitude higher, in most cases , in line with previous reports indicating that the heat stress response (HSR) in the male gametophyte is not robust as in vegetative tissues  in the MP_HS and MP_Hot/Cold datasets, respectively , was the most prominent, with a 23-fold increase , only increased threefold to fourfold during HS in the inflorescences  of wild-type Col-0 plants grown under control conditions or exposed to 3-day HS regime as in the MP_HS experiment . We show that five of six PSXs were upregulated in response to heat stress . Several miRNAs have been characterized as pollen-expressed under control conditions , which can impact mRNA degradation and translational repression. Similar to mRNA, lncRNAs can be targets of miRNAs and act as miRNA decoys, sequestering specific miRNAs , is a powerful method for identifying transcripts engaged with ribosomes and allows for the likelihood prediction of transcripts undergoing in vivo translation. In parallel with the RNAseq of the GP_HS experiment, sequencing of ribosome-protected fragments (RPF) was performed from the same in vitro HS experiment of in vitro germinated pollen  of actively elongating ribosomes. The PF P-site values were decomposed into the three different frames, to determine which frame is likely translated by the ribosome. One limitation of the XLOCs annotations is that no clear definition of exons has been established that allows the discrimination between coding and untranslated regions (UTRs). Therefore, we could not exploit the statistical significance of translational prediction of the RiboWave algorithm. However, visual inspection of the denoised PF P-site tracking allowed us to define a minimum number of five PF P-sites in the same frame as a proxy of an increased likelihood for translation. From the initial 45 XLOCs, 23 XLOCs passed this filter and could be considered as very likely translated  identified partial hits (50–96% identity) or full match hits (≥ 97% identity) with sequences of predicted proteins  for l16 (37%) of the genes, of which 92 (30%) are lncRNAs (Table S11). The proportion of blastx hits positives within the 75 RPFs XLOCs was 51%, and 60% (14 of 23) of the RPFs with the minimum number of five PF P-sites  for all 312 XLOCs (limited to XLOC_030571) and TCONS_00009618 (XLOC_006026), both pollen-specific and HS-induced expressed loci (Table S5).In Fig. XLOC_030571 clearly shows that only one reading frame (F1) is being translated, with the PF P-sites concentrated within the CDS along the transcript.XLOC_030751, annotated in Araport11 as unknown gene AT5G36985, is intronless with an ORF (DOC S3) coding for self-incompatibility protein homolog 25 (SPH25)  reported herein have gone unnoticed for such a long time is due, in part, for their expression being mostly restricted to pollen . Moreover, RNA extracted from the enriched isolated cells needs to be amplified to construct a library, which is biased toward transcripts with relatively high abundance, and transcripts with low copy number are often omitted  may account, at least in part, for its increased thermosensitivity, but also suggests that pollen has distinct requirements for coping with high temperatures. A major conceptual difference between pollen and sporophytic cells is that the latter can respond to HS by reducing metabolism into ‘survival mode,’ waiting for better growth conditions to resume growth or other physiological activities, whereas developing pollen and pollen tubes have a limiting window of time to properly mature and fertilize an ovule . PEPCK catalyzes the reversible decarboxylation of oxaloacetate to yield phosphoenolpyruvate (PEP) and CO2 at the expense of ATP. Since PEP is a precursor for either the glucose and shikimate biosynthesis pathways or to pyruvate, it is situated at an important crossroad in plant metabolism, lying between organic and amino acids, lipids and sugars Supplementary file1 (PDF 8293 kb)Supplementary file1 (PDF 50 kb)Supplementary file1 (PDF 304 kb)Supplementary file1 (DOCX 1357 kb)Supplementary file2 (XLSX 471 kb)Below is the link to the electronic supplementary material.


Existing literature informed that ageism might affect Internet use among older adults, further widening the digital divide among age groups. However, little empirical studies have been conducted on this topic. Our study aims to investigate (1) the current use of the Internet by gender, (2) the association between ageism and Internet use, and (3) potential gender differences. A cross-sectional data drawn from the 2016 Health and Retirement Study (HRS) was analyzed. Separate multiple regression analyses were conducted by gender to determine the varying impact of ageism on Internet use. We used two types of ageism (1) internal ageism (ingroup discrimination) and (2) external ageism  to observe each type’s contribution to Internet use. About half of the sample  reported using the Internet “daily,” while a quarter  responded, “never/not relevant.” No significant differences between gender were found in levels of Internet use, the rates of external ageism, or the degree of internal ageism. A higher level of ageism was associated with a lower level of Internet use. Interaction effects between age groups and ageism varied across gender: external ageism had interaction effects on men’s Internet use whereas internal ageism showed significant results for women. Our findings suggest that ageism may influence Internet use and its impact differs by gender. Gender-tailored intervention strategies should be developed to help older individuals to diminish the adverse effects of ageism on Internet use.


In this way, both O2 uptake and its distribution to the tissues are enhanced. Bilateral removal of the CB in humans leaves individuals unaware of hypoxemia, with complete abolition of the hypoxic ventilatory response.The pathogenic mechanisms underlying the symptomatology of coronavirus disease 2019 (COVID-19) patients are not well understood. An atypical and bewildering clinical manifestation found in many COVID-19 patients is that they exhibit severe hypoxemia, with arterial levels of oxygen .,2 sensitive glomus cells to support CB growth under sustained hypoxia.2-sensing is an intrinsic property of CB glomus cells, the functional responses of these cells are modulated by numerous auto- and paracrine signals generated within the organ. In this regard, a local renin–angiotensin system (RAS) and its principal components  have been described in the CB.The CB parenchyma is organized into clusters of cells called glomeruli. Each glomerulus is composed of 4–8 neuron-like glomus, or Type I, cells, which are in close contact with a network of fenestrated capillaries and are richly innervated by afferent sensory fibers of the petrosal ganglion. Glomus cells, the On = 4) as well as in mouse CB (data not shown). In addition to the histological characterization of CB ACE2 protein expression, we used qPCR to analyze the levels of mRNA ACE2 expression in our cohort of human CBs (n = 18). We found that the ACE2 mRNA levels are highly variable among all the CBs studied, without any clear correlation with donor age or gender (n = 15) whose medical records cited evidence of blood pressure before death. A nonsignificant trend toward a higher level of ACE2 mRNA expression was seen in CBs from donors with blood Type A with respect to Type 0. However, these comparisons have no statistical value due to the small sample size.To move this idea forward, we studied the immunohistochemical expression of ACE2 in adult human CB tissue using thin histological sections obtained from cadaveric donors available in the tissue bank of our institution. We showed that CB tissue expresses significant levels of ACE2. As expected, ACE2 staining appeared in blood vessels , as it ir gender . We did 2-sensing glomus cells could be potential targets for SARS-CoV-2 infection  and the European Research Council (ERC Advanced Grant PRJ201502629).None declared


The Rustenburg Layered Suite of the Bushveld Complex of South Africa is a vast layered accumulation of mafic and ultramafic rocks. It has long been regarded as a textbook result of fractional crystallization from a melt-dominated magma chamber. Here, we show that most units of the Rustenburg Layered Suite can be derived with thermodynamic models of crustal assimilation by komatiitic magma to form magmatic mushes without requiring the existence of a magma chamber. Ultramafic and mafic cumulate layers below the Upper and Upper Main Zone represent multiple crystal slurries produced by assimilation-batch crystallization in the upper and middle crust, whereas the chilled marginal rocks represent complementary supernatant liquids. Only the uppermost third formed via lower-crustal assimilation–fractional crystallization and evolved by fractional crystallization within a melt-rich pocket. Layered intrusions need not form in open magma chambers. Mineral deposits hitherto attributed to magma chamber processes might form in smaller intrusions of any geometric form, from mushy systems entirely lacking melt-dominated magma chambers. The Rustenburg Layered Suite of the Bushveld Complex, South Africa, has long been regarded as a textbook result of fractional crystallization from a melt-dominated magma chamber. Here, the authors find that the Rustenburg Layered Suite can be derived from crustal assimilation by komatiitic magma to form magmatic mushes without requiring the existence of a magma chamber by using thermodynamic models. Layered mafic intrusions, such as the iconic Rustenburg Layered Suite (RLS) of South Africa, have historically been considered to represent the solidified remnants of vast liquid-dominated reservoirs of magma called magma chambers where crystallization-differentiation has occurred by fractional crystallization4. However, an emerging consensus in igneous petrology views magmatic plumbing systems as being dominated by interconnected bodies of mush  extending from the base to top of the lithosphere and only rarely containing more than a few volume percent of liquid at ephemeral and isolated locations7. It is still problematic whether mafic–ultramafic layered intrusions represent shallow, large-scale melt pockets or crystal-dominated mush zones within transcrustal plumbing systems.Layered mafic intrusions represent portions of the plumbing systems of many large igneous provinces and are principal repositories of several critically important ore elements, including Cr, Ti, V, and the platinum-group elements (PGE)8, subject to recognition half a century later of the importance of crustal assimilation to result in the process of assimilation–fractional crystallization (AFC)9. The fundamental processes driving the evolution of magma composition in AFC are dissolution of host rock or xenoliths, accompanied by cooling and crystal growth, and the immediate removal of crystals from the possibility of continued reaction with the melt 4. This viewpoint has previously driven petrologists to search for the existence of the solidified remnants of such bodies in the rock record. A quintessential small example of closed-system fractional crystallization processes is the Skaergaard Intrusion of East Greenland11; however, the assumption that all large layered mafic intrusions must therefore also represent the solidified remnants of vast open chambers filled with melt is a reflexive model-driven extension of these ideas that has faced some recent challenges15.The paradigm of magmatic evolution by fractional crystallization has dominated igneous petrology since Bowen’s revolutionary advances a century agoelt Fig. . Given t16. As the proposed reason for fractional crystallization, the scenario of gravitational sinking of dense solids in static magmas8 does not universally hold true in magmatic systems, e.g., crystallizing phases may not separate from an evolved magma with high viscosity caused by crystallization and SiO2 enrichment. Additionally, heat exchange and crystallization in assimilation provide destabilizing buoyancy fluxes to drive forcefully disordered convection of mafic magma, where solids can be passively advected by vigorous convection instead of sinking17. In most turbulent komatiite flows, dense crystals can be carried by energetic eddies and remain in suspension without rapid separation18. Although equilibrium may not be attained at moderate temperatures, it is likely to occur rapidly in ultramafic magmas due to fast diffusions of elements in high-temperature, less-viscous liquids19. The success of a hypothetical dimensionless thermodynamic black box ABC process has been demonstrated numerous times, reproducing the observed compositions of ultramafic cumulate rocks and their supernatant magmatic liquids in intrusions both large and small22.A simple conceptual alternative to AFC is that of assimilation in conjunction with the textbook process of batch, or equilibrium, crystallization  RLS is the world’s largest layered mafic intrusive complex, containing ~600,000 km3 of mafic–ultramafic cumulates and extensive reserves of PGE, chromium, and vanadium that dominate global resources of these elements24. The RLS intruded the 2.6–2.3 Ga sedimentary Pretoria Group and 2.061 Ga felsic lavas of the Rooiberg Group  at upper-crustal levels (~0.06–0.24 GPa)25. In conjunction with the overlying Rashoop Granophyre and Lebowa Granite Suites , they together constitute the Bushveld Complex, comprising an enormous bimodal continental large igneous province in the Kaapvaal Craton  the fine-grained, noritic to peridotitic Marginal Zone (~100–750 m-thick), which flanks the other zones outside the main layered series and overlies a Basal Ultramafic Sequence (~750 m) encountered only in drill core beneath the other zones29; (2) discontinuous trough-like bodies of ultramafic Lower Zone (~800–1300 m) comprising harzburgite, orthopyroxenite, and minor dunite interlayers; (3) pyroxenitic Lower Critical Zone (~700–800 m) and noritic Upper Critical Zone (~500–1000 m), defined by the occurrence within both of them of prominent and laterally extensive chromitite and sulfide-bearing layers locally enriched in PGE; (4) gabbronoritic Main Zone (~3000–3400 m); and (5) uppermost ferrogabbroic-noritic and dioritic Upper Zone (~1700–2200 m) with abundant magnetite layers i)28. The Upper Zone is also subdivided into three subzones by the first appearances of magnetite (UZa), olivine (UZb), and apatite (UZc) i4. This is most clearly expressed in the Upper Critical Zone where variations in modal abundance within macrolayers such as the UG2 Unit result in juxtapositions of layers of anorthosite and norite with harzburgite, pyroxenite, and chromitite at scales of several meters12. However, zircon within these macrolayers has been shown in some cases not to have crystallized in a sequence younging upward through the stratigraphic column, an observation that has called into question the notion of a bottom-to-top aggrading crystal pile at the base of a long-lived magma chamber35. The formation of the upper portion of the RLS has been referred to as the differentiation stage 29. The Bushveld U-type magmas are compositionally similar to modern boninites formed by hydrous melting of metasomatized upper mantle40, but Barnes42 proposed a better analog in the siliceous high magnesium basalts derived from the crustal contamination of komatiites in Archean greenstone belts.Cumulate rocks in the complex can be subdivided into plagioclase-rich mafic units that have been postulated to be mafic crystallization products of tholeiitic magmas referred to as A-type magmas and ultramafic units that have been postulated to be products of high-MgO magmas referred to as U-type magmas41. The origins of the A-type magmas have had less attention than that of the U-type, with most investigators apparently assuming that they are commonplace tholeiitic basaltic magmas41 somehow derived from the upper mantle. Since the mantle does not directly produce tholeiites containing such low MgO contents, there must have been some processing of their parental magmas, though this process has not been clearly defined in the past1. B2 and B3 are also unconvincing parental magmas due to their partial cumulate characteristics and discrepant crystallization order compared to their interpreted cogenetic cumulates in the RLS25. The bulk composition of the lower portion of the RLS is too rich in compatible elements including Cr and the PGE to represent the composition of a liquid—it is necessarily regarded as being composed of cumulates deposited from larger volumes of through-going magma that are not presently exposed within the RLS43.The A-type magmas, thought to have contained ~7–8 wt% MgO, are tentatively correlated with fine-grained gabbronorites of the Marginal Zone where it abuts the Upper Critical Zone and Lower Main Zone, respectively, termed the B2 and B3 tholeiitic magmas , ascent rate as great as m/s and high liquidus temperature (>1550 °C) of komatiite18, its emplacement into cooler host rocks (~200 to 300 °C) approximates to forced turbulent convection during the early assimilation process, where solids are passively advected by chaotic flow and remain in suspension  that approximate to AFC as classically understood, presumably occurring during melt percolation through a complex lower-crustal magma reservoir that may have comprised multiple interconnected sill- and dike-like bodies largely composed of mush12. Major and trace element and Sr, Nd, and O isotopic compositions of the magmas and contaminants were estimated by comparison with upper-crustal and mid-crustal rocks exposed in the Pretoria Supergroup, Vredefort impact structure, and Limpopo Belt as documented in detail in Supplementary Table The parameters used in the models are provided in Supplementary Tables 25, following a previous Scenario 1 model for the UG2 pyroxenite of the Upper Critical Zone and complementary B1 magma12. After assimilation of 17.4% upper crust, Lower Zone dunite could form as an adcumulate comprising 4.5% trapped liquid; Lower Zone harzburgites require 22.5% assimilation and are modeled as mesocumulates comprising 17.8% trapped liquid, whereas Lower Zone and Lower Critical Zone pyroxenites could have formed after 27–34% assimilation of upper crust to leave a cumulate containing ~15% trapped liquid , while also failing to match observed whole-rock compositions.Ultramafic cumulates of the Lower Zone, Lower Critical Zone, and B1 marginal sill compositions were modeled under Scenario 1, assuming an upper-crustal assimilant at 0.2 GPa−2) than its current value (51 ± 6 mW m−2)56. The noritic Upper Critical Zone is xenolith rich, and has widely been regarded as an independent sill-like intrusion of progressive mixtures between B1 and B2/B3 magmas25. The similar crystallization sequence of Lower Main Zone also requires mixed parental magmas that intrude as crystal slurries from a deeper, staging reservoir after crustal assimilation24. We envision that their primitive parental komatiites experienced ABC assimilation  and cooled to ~1240–1250 °C in the middle crust to obtain the Bulk-1 compositions  after passage of the vast volumes of magma that produced the Main Zone, combined with muted temperature gradients, might have permitted efficient crystal separation during ongoing crustal assimilation17 i and εNd values of RLS i in B1, B2, and B3 marginal sills have been widely used to support assertions that these were samples of the U-type and A-type parental liquids of the RLS, but these observations are equally consistent with our proposition that the sills represent the liquid residue from deposition of the corresponding cumulates  in the RLS without apparent systematic changes is consistent with isotopic compositions of the proposed crustal assimilants  which contains some of the most 18O-depleted (<4.5‰) garnets in the global database60.We have modeled isotopic compositions of the cumulate rocks by tracking isotopic mass balances in the various mixtures of primary magma and assimilants according to the alphaMELTS models. The isotopic compositions observed in the RLS are compared with the results of our model of the transcrustal assimilation processes in Fig. RLS Fig.  is matchtes Fig. . The newnts Fig. , but notOur results show that the bulk of the RLS below Upper Main Zone, about 2/3 of its total thickness, appears to have been generated by either one-stage or at most, two-stage episodes of batch assimilation and crystallization and emplacement in their current locations as crystal mushes  and pyroxene–chromite cumulates are explicitly forbidden during fractional crystallization by the peritectic relations among olivine, orthopyroxene, and chromite but are entirely consistent with equilibrium phase relations.It is especially noteworthy that modal proportions of cumulus minerals in ultramafic cumulates such as olivine–chromite or olivine–orthopyroxene mixtures in layered mafic intrusions generally do not conform to the instantaneous cotectic modal proportions expected during fractional crystallization, positively requiring that many such cumulates were deposited and mechanically sorted into layers from polyphase suspensions that were broadly at internal equilibriumAs our modeling of the Upper and Upper Main Zone indicates, our goal here is not to argue that AFC and fractional crystallization alone are not valid petrogenetic processes, but instead to demonstrate that the idealized ABC concept represents a process sufficient to account for much of the spectrum of rock types observed in the world’s premier layered mafic intrusion, especially those world-class mineral deposits that are hosted by ultramafic macrolayers, and therefore cannot be ignored.29, Critical Zone chromite-bearing pyroxenites43, and Main Zone gabbronorites24 must have involved deposition of thick mushy layers, but these previous studies did not explore the implication that their mushy emplacement models are fundamentally inconsistent with AFC processes.Furthermore, in those cases where cumulates of contaminated magmas display modal proportions departing from expected cotectic proportions, some form of ABC must be accepted as having occurred. Indeed, a batch process is directly implied by several previous assertions that emplacement of the basal series dunites and harzburgites22. Rather, the compositions, textures, and modal variations in the Peridotite zone unequivocally require that a mixture of different mineral phases in cotectic proportions was mechanically sorted into layers including some that were nearly monomineralic within each larger macrolayer22. The ABC process does not require a large magma chamber to explain the sequence of rock types or to account for the lack of cyclicity; neither is the hypothetical existence of a magma chamber denied. We can consider layered mafic intrusions as products of numerous separate intrusive events in a long-lived magma column dominated by mushy zones punctuated by rare events when liquid-dominated magmas are transported and emplaced55. Macrolayers do not need to have formed in a younging sequence from bottom to top, although they might have. The hypothesis of mixing of fresh U-type magmas into resident A-type magmas of uncertain provenance to account for the sharp reversals in mineral assemblages that are associated with the major deposits of Cr and PGE35 is not necessary, and the apparently random sequence of mafic and ultramafic layers in the Upper Critical Zone can be regarded as the consequence of injection of crystal-rich magma batches that experienced different paths through the lithosphere either (a) in the observed stratigraphic sequence or (b) out of stratigraphic sequence—either scenario is consistent with the observed occurrence of alternating mafic and ultramafic layers. Emplacement of one mushy macrolayer into still-hot older cumulates that may or may not remain partially molten need not produce easily recognizable chilled margins71. On the other hand, the level at which these crystal mushes are emplaced and ponded is mostly but not exclusively sensitive to the depth in a crustal column at which they achieve neutral buoyancy. Notwithstanding the effects of local stress fields12, higher density ultramafic mixtures are likely to be emplaced beneath less-dense mafic zones, resulting in the general bottom-to-top sequence: ultramafic Lower Zone, pyroxenitic Lower Critical Zone, noritic Upper Critical Zone, gabbronoritic Main Zone, and noritic/dioritic Upper Zone below the pre-existing low-density volcaniclastic Rooiberg Group . In alphaMELTS, crustal material is incrementally added to the system, and an isenthalpic calculation employs entropy maximization to solve for thermodynamic equilibrium between silicate liquid and solid phases at constant pressure. Any resultant crystals can be equilibrated with or discarded from residual liquid , and the remaining system becomes the starting point for the next increment of assimilation. Following wholesale crustal assimilation at middle (0.45 GPa) and lower crust levels (1 GPa), further cooling of ascending magmas in the conduit was represented by an isobaric crystallization at 0.2 GPa and fayalite–magnetite–quartz solid oxygen buffer.Isenthalpic assimilation simulations were carried out using the AlphaMELTS software, version 1.9, and more information about these thermodynamic models can be found in the Supplementary Tables where we provide copies of the melts files and environment files used. Model results appear in the Supplementary Data 54. Because slight differences in the Gibbs free energy among various candidate model pyroxenes may confuse the choice of the second pyroxene after orthopyroxene, we corrected the pyroxene/melt partition coefficients when the algorithm improperly terms a high-Ca clinopyroxene as the second orthopyroxene in the B3-Gn (Main Zone) lineage.Variable rare-earth element partition coefficients of clinopyroxene/melt and feldspar/melt were calculated based on lattice strain theory, while constant partition coefficients for remaining elements and melt-solid pairs were taken from a comprehensive review87Sr/86Sr)i and εNd of mantle-derived magma are calculated as 0.7016 and 3.97, respectively. The δ18O of an uncontaminated mantle-derived magma is widely considered as 5.7‰ in light of the global range of 5.4–6.0‰ in fresh MORB glasses60. (87Sr/86Sr)i and εNd values of middle crust and lower crust are the averages of extensive collected data in this region  than the averages of overlying volcanites (7.36‰), granophyres (6.6‰), and granites (7.35‰), and a moderate value (9.6‰) was set for the upper-crustal materials  assuming a possible greater contribution from δ18O-enriched metapelites. The proposed lower crust has moderate Mg# and SiO2/Al2O3 ratios, corresponding to the features of intermediate-type lower-crustal granulite xenoliths that has an average δ18O of 9.2‰75.Pretoria Group rocks have the higher Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1


This special report was developed to communicate policy and procedures for free-standing acute inpatient rehabilitation hospitals (AIRHs) to protect patients and healthcare personnel and to prevent further spread of severe acute respiratory syndrome coronavirus 2. The recommended policies were developed in conjunction with the New Mexico Department of Health and hospital leadership. As we attain additional knowledge and experience during this pandemic, suggestions of best practice will continue to evolve for AIRHs. The authors encourage readers to work with local regulatory officials to ensure regulatory compliance as well as respect of the availability of local resources. This special report communicates potential policy and procedure modifications for free-standing acute inpatient rehabilitation hospitals in response to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)/coronavirus disease (COVID-19) pandemic. The goal of the communication is to help protect patients and healthcare personnel and to prevent further spread of SARS-CoV-2. The recommendations were developed in conjunction with the New Mexico Department of Health and hospital leadership. A comprehensive response policy to SARS-CoV-2/COVID-19 should include facility changes, enhanced personal protective equipment policy, enhanced admission policy and enhanced surveillance testing policy. In the setting of rapidly changing state and federal guidelines, hospital staff should work with state officials to ensure that potential policy and procedure modifications are in line with state and federal recommendations. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is the infectious source for coronavirus disease COVID-19) was first reported on 31 December 2019 9 was fir. In BernThe rapid spread and sheer number of cases have exacted a global and national toll on the healthcare system as healthcare professionals balance the need for continued patient care with the need to control the spread of the virus . This haSupplementary material to this article and can be found online [Our free-standing acute inpatient rehabilitation facility is in a unique position because it is one of only four such facilities in the state of New Mexico capable of providing complex acute inpatient rehabilitation services. Our rehabilitation hospital is the only hospital in New Mexico accredited by the Commission on Accreditation of Rehabilitation Facilities for 3 years in six programs: Comprehensive Integrated Inpatient Rehabilitation Program, Inpatient Brain Injury Program, Inpatient and Outpatient Spinal Cord System of Care, Inpatient Stroke Specialty Program, Outpatient Medical Rehabilitation Program, and Outpatient Medical Rehabilitation Program (Children and Adolescents). The hospital has 265 employees with 43 contractors. There were 62 licensed acute inpatient rehabilitation beds and 1470 inpatient admissions in 2019. The hospital provides a full continuum of inpatient and outpatient rehabilitation care, including physical therapy, occupational therapy, speech and language pathology, rehabilitation nursing and case management services. The inpatient program is designed for patients with a variety of rehabilitation needs, offering general inpatient medical rehabilitation and three specialized programs: brain injury rehabilitation, stroke rehabilitation and spinal cord injury rehabilitation. Details of the program are supplied as d online .On 6 July 2020, senior hospital leadership was informed of a patient who discharged on 3 July 2020 then tested positive for COVID-19 two days after being discharged. In response to this notification, all patients and staff were tested that same week as part of a swift and organized response. Testing resulted in identification of three patients and four staff members with positive COVID-19 tests. Given the acute inpatient rehabilitation population at our facility  is at high risk of being affected by respiratory pathogens such as COVID-19, a comprehensive assessment was made with resulting action plans .Our hospital has an established infection prevention and control program. It became clear, however, that new measures were needed in the setting of this unique pandemic. The medical director, in conjunction with state officials from the Department of Health and senior hospital leadership, developed new procedures and policies as detailed below.The goal of the SARS-CoV-2/COVID-19 specific policies and procedures is to protect patients and healthcare personnel and to prevent further spread of the virus. These policies and procedures may be of benefit to other free-standing acute inpatient rehabilitation facilities.Identify SARS-CoV-2 exposuresContain SARS-CoV-2 exposuresSafely maintain average daily censusThe specific aims of the program are as follows:The efficient and timely identification of SARS-CoV-2 exposures mitigates risk of an outbreak across our hospital system and protects the vulnerable patient population. Containment limits exposures to a single unit, allowing for continued patient flow in other units, ultimately allowing for uninterrupted admission and the ability to maintain the average census. This enables ongoing, high-level rehabilitation services while also allowing admission from overwhelmed acute care hospitals.To help prevent or reduce airborne transfer of infectious organisms and viruses through airborne transmission, all hospital filters were changed to minimum efficiency reporting value-13 (MERV-13). The MERV-13 filters are reported to filter 90% of particles in the 3–10 μm range, 85% of particles in the 1–3 μm range and 50% of particles in the range 0.3–1 μm range .Enhance personal protective equipment (PPE)  were delivered and deployed to hospital staff. This enhanced PPE protocol was made mandatory; the use of cloth, homemade masks was initially discouraged and later banned.All potential patients must be free of symptoms typically associated with SARS-CoV-2 for 7 days before admission, unless attributed to other etiologies All potential patients not previously identified as COVID-19 positive are required to have at least one negative test within 72 h before admissionPatients previously identified as COVID-19 positive require two negative tests >24 h apart before admissionAll newly admitted patients are tested upon admission regardless of the last COVID-19 testAll admitted patients tested weeklyWeekly rotating surveillance testing of 25% hospital staffCohorts established with designated units, rehabilitation gym space, nursing, therapy and ancillary staff Each cohort consisting of 31 patient rooms (double occupancy) with dedicated nursing, therapy (assigned to unit) and ancillary staff (assigned to cohort)Nursing, therapy and ancillary staff cannot visit/aid units not in designated cohortSocial distancing procedures  are enfoPatients identified as positive through the admission testing or weekly surveillance testing process will be immediately transferred to acute care hospital for medical triage and treatmentAffected unit would be quarantined for at least 2 weeks: — Comingling between adjacent units in cohort will cease immediately and continue to be prohibited until such time as the quarantine is lifted — No new patients admitted to affected unit and no patients will be transferred out of affected unit other than to discharge Designated staff responsible for cohort in which patient was assigned will undergo immediate testing and subsequent weekly testing until no new positive results are detectedThe goal of the developed policy and procedures is to protect patients and healthcare personnel and to prevent further spread of SARS-CoV-2. The policies were developed in conjunction with an infectious disease epidemiologist at the New Mexico Department of Health. The authors feel that these processes and procedures may be of benefit to other free-standing acute inpatient rehabilitation facilities.These suggestions for updated facility policies in response to the SARS-CoV-2/COVID-19 pandemic are in line with the experience of other facilities . As we aThe development of our hospital's policies and procedures follows current recommendations and prior experience with respiratory pandemics. In the setting of rapidly changing state and federal guidelines we also worked with New Mexico state officials to ensure our recommendations were in line with state and federal recommendations. The authors encourage readers to work with local regulatory officials to ensure any polices or procedures developed are within regulatory compliance, respects the availability of local resources and addresses specific local conditions.There will be a need to develop detailed federal and national guidelines for acute inpatient rehabilitation hospitals in response to SARS-CoV-2/COVID-19 pandemic and future viral pandemics. These guidelines will ensure rehabilitation hospital policies and procedures are within regulatory compliance, respect the availability of national and local resources and appropriately address the evolving pandemic. In the absence of such guidelines, rehabilitation hospitals will have to work with state officials to develop guidelines in response to the SARS-CoV-2/COVID-19 pandemic.Policy and procedures should be developed for acute inpatient rehabilitation hospitals to protect patients and healthcare personnel and to prevent further spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)/coronavirus disease (COVID-19).In the setting of rapidly changing state and federal guidelines hospital staff should work with state officials to ensure recommendations are in line with state and federal recommendations.A comprehensive response policy to SARS-CoV-2/COVID-19 should include facility changes, enhanced personal protective equipment policy, enhanced admission policy and enhanced surveillance testing policy.