We found the combined treatment to be safe with grade 3 toxicity reported in three patients. As the best overall response, four patients had a partial response, eight had stable disease and five had progressive disease. Median overall survival time was still not reached after a median follow‐up of 26.5 months and 10/15 patients with programmed death‐ligand 1 negative tumours were alive >18 months after the start of the study treatment. ctDNA was detectable at baseline in 11 patients. A rapid decline in ctDNA to <30% of baseline levels was seen in three patients, two of which were radiographic responders and one was considered clinically benefiting from therapy for almost 1 year.The introduction of immune checkpoint inhibitors has transformed the treatment landscape of metastatic non‐small cell lung cancer. However, challenges remain to increase the fraction of patients achieving durable clinical responses to these drugs and to help monitor the treatment effect. In this phase II trial, we investigated the toxicity, systemic responses and circulating tumour DNA responses in patients ( Immunotherapy has transformed the treatment landscape of advanced non‐small cell lung cancer. However, challenges remain to increase the fraction of patients achieving durable responses to these drugs and to help monitor the treatment effect. In this clinical trial, we investigated the tolerability, efficacy and circulating tumour DNA responses in patients with metastatic lung cancer treated with atezolizumab combined with radiotherapy. AFallele frequencyALKanaplastic lymphoma kinaseBEDbiologically effective dosecfDNAcell‐free DNAcfNAcell‐free nucleic acidCNVcopy number variantsCRcomplete responseCTCAEcommon terminology criteria for adverse eventsctDNAcirculating tumour DNAEDTAethylenediaminetetraacetic acidEGFRepidermal growth factor receptorERBB2erythroblastic oncogene BFFPEformalin‐fixed paraffin‐embeddedGTVgross tumour volumeICIimmune checkpoint inhibitorindelsinsertion–deletion mutationsKRASKirsten rat sarcoma viral oncogene homologLODlimit of detectionMETmesenchymal–epithelial transition factorNEnot evaluablengnanogramNGSnext‐generation sequencingNSCLCnon‐small‐cell lung cancerORRobjective response rateOSoverall survivalPD‐1programmed cell death protein‐1PD‐L1programmed death‐ligand 1PFSprogression‐free survivalPRpartial responsePSperformance statusPTVplanning target volumeRTradiotherapySBRTstereotactic body radiotherapySDstable diseaseSNVsingle nucleotide variants1in situ vaccine [Lung cancer is one of the main contributors to the global cancer burden with an estimated 2.21 million new cases and 1.79 million deaths worldwide in 2020 . Unfortu vaccine . Further vaccine , 11. All vaccine . Several vaccine , 15, 16.Assessment of therapeutic efficacy in patients with NSCLC ismainly based on radiographic scans. This evaluation method has its limitations when monitoring immunotherapy response. Checkpoint inhibitors sometimes cause a transient increase in tumour size due to infiltration of immune cells, pseudoprogression and response to ICIs on CT scans can be delayed , 18, 19.Cell‐free DNA (cfDNA) are degraded fragments of cellular DNA, usually 140–200 base pairs in length, found in the circulation of both healthy and diseased individuals , 22. ctDIn this study, we investigated the combination of a PD‐L1 inhibitor, atezolizumab and radiotherapy in chemotherapy‐pretreated patients with advanced NSCLC. We here present the clinical outcomes, including therapeutic responses and adverse events, as well as mutations detected in ctDNA, changes in ctDNA during treatment and how they relate to therapy responses at an individual level.22.1recist 1.1) and a tumour lesion suitable for stereotactic body radiotherapy. Key exclusion criteria were significant cardiac, pulmonary or other medical illness that would limit activity or survival, previous treatment with a PD‐1/PD‐L1 inhibitor, a history of drug‐induced or idiopathic pneumonitis, active/untreated brain metastases and medical conditions requiring > 30 mg·day−1 of prednisone or equivalent. Patients with EGFR mutation or ALK translocation were eligible if they had previously been treated with a tyrosine kinase inhibitor.The Combinatory ImmunoTherapy‐1 (ComIT‐1) trial is a multi‐center phase II trial conducted at three university hospitals in Norway. In this study, we included patients with advanced NSCLC (stages III–IV) previously treated with a platinum doublet. Eligible patients were > 18 years of age with an ECOG performance status score of 0 or 1, adequate organ and hematologic functions, measurable disease according to Response Evaluation Criteria in Solid Tumours version 1.1 (2.23 in volume as determined by gross tumour volume (GTV), between the first and second infusion of atezolizumab. Metastatic lesions were preferred for irradiation, and at least one tumour lesion should not be irradiated to act as a target lesion for RECIST evaluation. The primary endpoint was toxicity. Secondary endpoints included response rates, progression‐free survival (PFS), duration of response, OS, abscopal effects and quality of life. Exploratory endpoints included immunological response, tumour evolution, dynamics in the tumour microenvironment and biomarkers of clinical response. The study was conducted in accordance with the ICH E6 guidelines for Good Clinical Practice, the principles of the Declaration of Helsinki [www.clinicaltrials.gov .Patients received the PD‐L1 inhibitor atezolizumab concomitant with SBRT. Atezolizumab was administered at a fixed dose of 1200 mg intravenously every 3 weeks for a maximum of 2 years until intolerable toxicity or no clinical benefit as judged by the investigator. SBRT 6 Gy × 3, fractions 1 day apart, was given towards one or two tumour lesions, minimum 2 cmHelsinki  and was 2.3recist 1.1 [ctcae) version 4.0 .Tumour evaluation with CT scans was performed at baseline and then every 9 weeks for the first 6 months, thereafter every 12 weeks. Patients underwent supplemental MRI and 18F‐FDG‐PET‐scans if clinically indicated. Tumour response was assessed according to cist 1.1 , includi2.4g for 10 min within 1 h of collection, immediately aliquoted and conserved at −80 °C. Total cfNA were isolated from 2 mL EDTA‐plasma using MagMAX Cell‐Free Total Nucleic Acid Isolation Kit , according to the manufacturer's recommendations. The elution volume was 20 𝜇L. Targeted Sequencing Libraries were prepared manually using the Oncomine™ Pan‐Cancer Cell‐Free Assay. Inputs were 1.46–77.38 ng cfNA (median 7.69 ng). Libraries were quantified with qPCR, amplified and loaded on Ion 540 chips by Ion Chef and sequenced on the Ion GeneStudio™ S5 system (Thermo Fisher Scientific). Output data were analyzed using the ion reporter software (Thermo Fisher Scientific). We considered all single nucleotide variants (SNVs) and indels with an allele frequency (AF) ≥ 0.1 at baseline as significant provided that the AF surpassed the limit of detection (LOD) calculated using the given DNA input. Copy number variants (CNVs) had to exceed 1.4× fold change to be reported. In case of multiple mutations, we used the one with the highest AF before the start of treatment for longitudinal monitoring.Blood samples were collected at baseline, during radiotherapy, at every evaluation time point and at disease progression. Three 10 mL‐EDTA tubes of peripheral blood were taken, and plasma was separated by centrifugation at 1000 To confirm that genetic alterations detected in blood originated from tumour tissue, we did a genomic analysis of formalin‐fixed paraffin‐embedded (FFPE) tissue samples collected before the start of study treatment or at the time of diagnosis if no dedicated pre‐treatment biopsy was available. Targeted libraries were prepared on the Ion Chef from 20 ng of tumour DNA with the Ion AmpliSeq™Cancer Hotspot Panel v2, quantified with qPCR, amplified and loaded on Ion 540 chips by Ion Chef and sequenced on the Ion GeneStudio™ S5 system (Thermo Fisher Scientific). Output data were analyzed using the Ion Reporter software (Thermo Fisher Scientific).33.1EGFR mutations with progression on EGFR inhibitors and platinum‐based chemotherapy. Fifteen patients were PD‐L1 negative and 13 received atezolizumab as second‐line treatment.We enrolled 21 patients; 13 men and 8 women, with a mean age of 61.7 years, in the trial between September 2018 and February 2020. (Table Lung tumours were selected for SBRT in 16 cases. Only one patient received SBRT for more than one lesion. Treatment details regarding radiotherapy can be found in Table 3.2n = 6), flu‐like symptoms (n = 5) and elevated liver enzymes . Of special interest, pneumonitis occurred in three patients, of which two had received SBRT to lung lesions. In both these patients the pneumonitis was grade 1. Except for these cases, adverse events were mainly systemic in nature and not confined to irradiated areas. A summary of adverse events and relevant treatment‐related toxic effects are shown in Tables Fifteen patients (71%) experienced adverse events of any grade possibly or definitely related to treatment. Grade 3 treatment‐related toxicity was reported in three patients; one colitis, one pneumonitis and one skin toxicity. The patient with colitis had to permanently stop treatment due to a recurrent grade 3 event. For the patients with grade 3 pneumonitis and grade 3 skin toxicity, oral prednisolone was administered and atezolizumab was temporarily held for 8 and 3 weeks respectively, until symptoms had recovered to grade 1. In both cases, atezolizumab was successfully restarted. There were no grade 4 or 5 events. The most common adverse events were skin rash (3.3recist v1.1. Two of these patients (patients 19 and 21) had a rapid symptomatic deterioration probably due to disease progression though not radiographically confirmed. The other two (patients 3 and 14) were considered to have a prolonged (>18 months) clinical utility from treatment without the progression of non‐target lesions or the appearance of new lesions. However, they were not evaluable after radiotherapy, patient 3 due to pneumonitis making the assessment of the target lesion impossible, and patient 14 because both measurable lesions in the hip and pelvis were irradiated shortly after the first infusion of atezolizumab. In general, there was a trend towards two distinctive patterns of response among patients, either a long‐lasting clinical benefit or a rapid worsening of the disease. Eight patients were on treatment for more than 12 months, and three of them were treated beyond radiological progression. In contrast, seven patients had a time to treatment discontinuation < 3 months. Clinical outcomes for the individual patients are shown in Fig. At the cut‐off date for analysis (6 September 2021), the median follow‐up time was 26.5 months (range 17.6–35.5). Two patients were still receiving treatment. A median of nine doses (range: 1–35) of atezolizumab was administered and five patients completed 2 years of treatment. Median PFS was 4.3 months (95% CI 2.2–8.7). Median OS time was still not reached at the data cut‐off. As the best overall response, four patients had a PR, eight had stable disease (SD) and five had progressive disease (PD). Among the four patients achieving av PR, the median duration of response was 17.8 months. Four patients were not evaluable (NE) according to The efficacy endpoints focused on the systemic responses, and irradiated lesions were excluded as target lesions. However, when reviewing the treatment effect on irradiated lesions, there were no patients whose non‐irradiated lesions responded better than the irradiated ones. All four patients with PR as the best overall response also had a significant reduction of the irradiated tumours, in two cases leading to a complete radiologic disappearance of the tumour.3.4The 15 participating patients with PD‐L1 negative tumours had a median of 10 infusions of atezolizumab. As the best overall response, two patients achieved a partial response and seven had SD, creating an objective response rate (ORR) of 13% and a disease control rate of 60%. Median PFS was 4.1 months. Six PD‐L1 negative patients were on treatment and considered clinically benefitting from therapy for more than 1 year, although three of them were treated beyond radiologic progression. Median OS time was still not reached at the data cut‐off, but 10 out of the 15 patients were alive more than 18 months after the start of the study treatment.3.5TP53 (five patients) followed by KRAS (three patients). Three patients had more than one detectable variant. No fusions were detected. From 8 of the 11 patients with cancer‐associated genetic alterations in plasma at baseline, we had FFPE tissue samples available for genomic analysis. In seven of these cases identical mutations were also found in tumour tissue. One patient had a TP53 mutation and a MET amplification was detected in blood, but not in tumour tissue.Baseline samples for ctDNA analysis were available from 19 patients. Lung cancer‐associated somatic mutations were identified in 11 of these Fig. . The gen3.6KRAS detected in blood at baseline . These patients had in general poor response to atezolizumab with only one patient achieving a partial response and four out of six patients experiencing a rapid progression. In contrast, of the three patients with KRAS mutations two were considered partial responders while the third had a prolonged clinical benefit although not evaluable according to recist v1.1.Six patients had targetable driver mutations other than 3.7recist v1.1. Figure We examined the relationship between ctDNA responses and radiographic responses for each patient during treatment. ctDNA was quantified using the allele frequencies of mutant tumour‐derived DNA. Of the 11 patients with detectable ctDNA at baseline, one did not have additional blood samples suitable for ctDNA analysis and one did not have measurable disease according to In the responder group, two out of three patients had a rapid decline in ctDNA to <30% of baseline levels within 2 weeks. In both patients, the ctDNA continued to decrease until undetectable levels at the second blood draw 9 weeks after the start of treatment. The third radiographic responder (patient 09) had a slight increase in ctDNA during radiotherapy and did not experience a significant drop in ctDNA. Patients without a radiographic response showed a more variable ctDNA trend. Only one of these six patients had a drop in ctDNA level to < 30% of baseline at 2 weeks. Four patients in this group had minor changes in ctDNA levels at the first checkpoint (< 30% change from baseline). One patient did not have an additional plasma sample before week 9, when the ctDNA had increased dramatically.When following the sequential changes in ctDNA and radiographic tumour burden for each patient during treatment Fig. , there wMET) was quite stable, the second (KRAS) reached undetectable levels during treatment. In patient 15, the pre‐treatment alterations in plasma affecting the TP53‐ and MET‐genes were detected at similar quantities during radiotherapy but were not detectable at later timepoints when the tumour progressed. A potential explanation for this mismatch could be the development of treatment‐resistant clones harbouring other genetic alterations [Patient 09 was registered as a responder on CT scans after 11 months. This patient had two detectable cancer‐associated mutations in plasma at baseline, and while the level of the one with the highest AF  underwent treatment with atezolizumab for more than 12 months and 10 (67%) were alive more than 18 months after the start of the study treatment. These numbers are encouraging, considering subgroup analysis from the phase III OAK trial which showed that atezolizumab was superior to docetaxel in previously treated NSCLC patients [0% underwNevertheless, a significant proportion of our patients did not respond to the study treatment. One reason might be that the SBRT schedule was not ideal. The optimal radiation dose, fractionation and timing to generate an immune response and enhance the effect of combining radiotherapy with immunotherapy remain to be defined. Preclinical models suggest that prior radiotherapy upregulates PD‐L1 expression and thus facilitates later administration of ICIs . In a meIn our trial, radiotherapy was given as SBRT, but with significantly lower doses than standard curative stereotactic radiotherapy. This radiotherapy dosing was chosen to induce immunogenic cell death while being well tolerated and was in accordance with other studies testing the combination of immunotherapy and radiation . IncreasMultisite SBRT represents another interesting approach to optimize the combined treatment of radiotherapy and immune checkpoint inhibitors . SBRT diIn advanced NSCLC, there is a need for new predictive biomarkers to better identify patients who will benefit from immune checkpoint blockade and to help monitor the treatment effect. A promising tool in this setting is ctDNA. Previous studies have demonstrated a strong correlation between longitudinal ctDNA changes, radiographic responses and clinical outcomes in stage IV NSCLC patients treated with ICIs , 29, 30.A potential advantage of ctDNA is its rapid kinetics. Several studies have reported that the ctDNA response considerably precedes the radiological response in NSCLC patients treated with immunotherapy , 25, 27.Some studies have reported a transient flare‐up of AF within the first 14 days after the start of immunotherapy in a subgroup of patients, possibly due to the increased release of DNA from dying tumour cells , 29, 38.EGFR, ERBB2 and MET) had a poor treatment response, while patients with a KRAS mutation detected in plasma‐derived more benefit. This is well documented in tissue biopsies and the same response pattern is expected to be found with liquid biopsies [The subtype of mutation detected in blood seemed to impact the response to atezolizumab in our trial. In accordance with previous reports, we found that patients with a targetable driver mutation (biopsies , 39, 40.A major limitation is the small and heterogeneous study population and the non‐randomized design, which makes it difficult to draw firm conclusions. To capture the role of SBRT in the combined approach with respect to improving PFS and response rates, a larger trial containing a comparison group receiving ICI alone is needed. Furthermore, pre‐treatment ctDNA was detected in only 11 of the 21 enrolled patients and due to incomplete tissue genotyping data, we were not able to confirm the concordance between mutations detected in plasma and mutations in tissue for all patients. In theory, mutations identified in plasma could represent contamination from germline variants and clonal hematopoietic mutations and not be attributed to tumour cells. We could have reduced this risk by doing paired peripheral blood mononuclear cell NGS or by excluding mutations with AF > 20% in all patient samples . In one 5In conclusion, concurrent atezolizumab and SBRT were safely administered in metastatic NSCLC patients, yielding encouraging results in a subset of patients, including those with PD‐L1 negative tumours. Additional trials are needed to evaluate the optimal radiation dose, fractionation and sequence to increase systemic response rates. ctDNA holds potential as a dynamic biomarker in lung cancer patients receiving immunotherapy, however, questions remain concerning the robustness and practical implementation.Henrik Horndalsveen: Advisory board: Janssen. Honoraria: AstraZeneca, Pfizer, Roche. Bjørn Henning Grønberg: Advisory board: AstraZeneca, BMS, Debiopharm, Eli Lilly, Janssen, MSD, Novartis, Pfizer, Roche, Takeda. Honoraria: AstraZeneca, Bayer, BMS, Bohringer Ingelheim, Debiopharm, Eli Lilly, MSD, Novartis, Pfizer, Pierre Fabre, Roche, Sanofi, Takeda. Research funding: AstraZeneca, Roche. Spouse: Employee and shareholder of Eli Lilly and Company. Tarje Onsøien Halvorsen: Honoraria: AstraZeneca, MSD, Pierre Fabre, Pfizer. Travel support: AstraZeneca, MSD. Vilde Drageset Haakensen: Advisory boards: Novartis, Astra Zeneca, Pfizer. Honoraria: BMS, Astra Zeneca, Takeda. Åslaug Helland: Financial support and/or study drug from AstraZeneca, Roche, Novartis, Incyte, Eli Lilly, Ultimovacs and BMS, in association with clinical studies. Adv board: AstraZeneca, BMS, Janssen, MSD, Pfizer, Roche, Takeda, Sanofi, Bayer, Abbvie. The remaining authors declare that they have no competing interests.HH: Clinical investigator, analysis of data, drafting the manuscript. TNA: Plasma and tissue sequencing, analysis of data. AMD: Plasma and tissue sequencing, analysis of data. LVR: Clinical investigator. CR: Analysis of data. NH: Clinical investigator. BHG: Clinical investigator. TOH: Clinical investigator. VDH: Clinical investigator. ÅKÖ: Analysis and visualization of data. MMB: Clinical investigator. ÅH: Principal investigator, attract funding, writing protocol, analysis of data. All authors revised and approved the final version of the manuscript.https://publons.com/publon/10.1002/1878‐0261.13330.The peer review history for this article is available at Fig. S1. Plasma levels of ctDNA and overall radiographic tumour burden during treatment for each patient.Click here for additional data file.


Reliably diagnosing autism spectrum disorders (ASD) in adulthood poses a challenge to clinicians due to the absence of specific diagnostic markers. This study investigated the potential of interpersonal synchrony (IPS), which has been found to be reduced in ASD, to augment the diagnostic process. IPS was objectively assessed in videos of diagnostic interviews in a representative referral population from two specialized autism outpatient clinics. In contrast to the current screening tools that could not reliably differentiate, we found a significant reduction of IPS in interactions with individuals later diagnosed with ASD (n = 16) as opposed to those not receiving a diagnosis (n = 23). While these findings need to be validated in larger samples, they nevertheless underline the potential of digitally-enhanced diagnostic processes for ASD.The online version contains supplementary material available at 10.1007/s10803-021-05194-3. Nonverbal abnormalities in the social domain have been identified as a key impairment in autism spectrum disorder  consisting of individuals who received a diagnosis of F84.5 , F84.1 , or F84.0  according to ICD-10  consisting of individuals for whom any F84 diagnosis (including F84.9) was ruled out.Participants were drawn from a representative clinical referral population of adults referred to autism diagnostics to the specialised autism outpatient clinics of the University Hospitals of Munich and Cologne. All participants were assessed while undergoing real-life diagnostic procedures and had been referred to the specialist clinics by medical consultants (psychiatry and neurology) on the basis of suspicion of a possible ASD diagnosis due to reported social-emotional symptoms. Diagnostic procedures have been conducted in accordance to German guidelines for diagnosis of ASD in adulthood  =  − 0.437, p = 0.664), motor difficulties  and age . Descriptive statistics can be found in Table The groups were frequency-matched with respect to IQ  with a frame rate of 25 fps. The interviews were conducted by eight different diagnosticians (including JK). Subsequently, patients underwent several hours of analysis of medical history, former parental/caretaker interviews and neuropsychological assessments  to compute a total ROI per person.Videos were analyzed post-hoc with Motion Energy Analysis Version 3.10 . To this end n = 1,000 surrogate synchronies were computed  by aligning time series of participants in random order who never actually interacted with one another  within every ROI . This makes the synchrony in the body ROI more difficult to interpret. For reasons of completion we have reported these, along with the total ROI in the supplementary materials. We therefore report the head ROI and have good reason to do so: In previous research on IPS, head synchrony has been associated with the overall outcome of psychotherapy .To ascertain that the method yielded valid IPS measurements, we compared mean IPS with surrogate data across participants using the Mann–Whitney test, as a nonparametric alternative for independent samples t-test  = − 0.326, p = 0.746, dCohen =  − 0.106), suggesting similar amounts of mean overall head movement in both groups.To rule out the overall amount of movement as a confounding factor for potential differences in IPS, we examined differences in average movement quantity per dyad across groups. We performed an independent samples t-test. No significant differences were found between groups  = − 2.068, p = 0.023, one-tailed, d = − 0.673). IPS was significantly lower in interviews with ASD+ patients than with ASD− patients not fulfilling diagnostic criteria.To investigate group differences in interpersonal synchrony, we performed an independent samples t-test comparing ASD+ and ASD− . We found a significant difference of IPS head synchrony between the ASD + and the ASD- group  = − 1.065, p = 0.294, d = − 0.347; see Table U = 239.5, p = 0.116, d = 0.302; see Table We conducted an independent samples t-test for the included standard screening instruments for autism traits (AQ) and empathy (EQ) and subsequently correlated them with the IPS values within groups. We found no significant group differences for both Below is the link to the electronic supplementary material.


Helicobacter pylorigastritis can cause serious adverse effects in the short and long term. I‐scan optical enhancement (OE) has a potential role to distinguish areas of infected mucosa and allow for targeted biopsy. It improves visual contrast and mucosal pattern characterization. The work aims to determine if the diagnostic yield of the CLOtest could be improved by using endoscopic I‐scan OE technology for targeted gastric biopsy sampling.H. pylori infection diagnosed by C13 UBT at Nizwa General Hospital from March 2021 to January 2022. The patients underwent a careful examination by nonmagnifying upper endoscopy and I‐scan OE 3 moods, then randomly allocated into two groups. Group A: nontargeted double biopsies from the antrum and mid corpus. Group B: I‐scan OE‐directed targeted biopsy from abnormal mucosal patterns. The biopsy specimens were inoculated into CLOtest kits; the reading time of the positive results was at 1, 4, and 24 h.A prospective study recruited 112 adult patients with active p = 0.501). One‐hour CLOtest was positive in 78.5% of the patients in group B compared to 60.7% in group A (p = 0.047), while group A had a significantly more positive CLOtest at 24 h.Group B had a 92.8% positive CLOtest compared to 89.3% in group A (Sampling a targeted gastric biopsy with the aid of I‐scan ‐OE for CLOtest significantly hastens the positive reading time with high sensitivity. It allows for mucosal identification for targeted biopsy sampling.H. pylori organisms by RUT.H. pylori infection may be missed if not present in random biopsy samples from normal‐looking mucosa with a resultant increase of false‐negative results.Several gastric mucosal findings have a high probability of harboring H. pylori gastritis.H. pylori infection‐related abnormal endoscopic features with higher diagnostic accuracy.I‐scan technology is a digital, image‐enhanced endoscopic (IEE) technology by PENTAX Medical. It enhances the endoscopic detection, demarcation, and characterization of the mucosal and vascular patterns by providing a clear, enhanced image and detailed topography. It can detect more subtle mucosal abnormalities,H. pylori infection.Pentax's new endoscopic platform integrated the second‐generation “I‐scan optical enhancement” (OE). It incorporates an optical filter with improved visual contrast and provides extra information through improving vessel and mucosal pattern characterization.2Aprospective study included 112 adult patients referred for upper endoscopy for variable indications. They all had untreated active H. pylori infection diagnosed by a C13 urea breath test (UBT). The study was carried out from March 2021 to January 2022 at Nizwa General Hospital, Ministry of Health, Sultanate of Oman. Exclusion criteria: history of any of the following; recent use of antibiotics, bismuth‐containing compounds (2 weeks), and PPIs (4 weeks), recent or acute upper gastrointestinal bleeding, gastric surgery, IM, atrophic gastritis, PUD, liver cirrhosis, portal hypertensive gastropathy, alcohol consumption, other causes of chronic gastritis , infection other than H. pylori, achlorhydria, gastric cancer, coagulopathy, or patients without endoscopic features or signs of gastritis.The recruited patients were assessed clinically and biochemically, then examined by upper endoscopy, initially with HD‐WLE (high definition WLE), followed by I‐scan OE 3 moods successively. The patients were randomly distributed (before endoscopy) in a 1:1 ratio  into;Group A : included 56 patients; two samples were taken, one from the antrum on the greater curvature, approximately 3 cm from the pyloric ring, avoiding areas of ulceration and obvious IM, and one from normal‐appearing corpus near mid‐body on the greater curvature.Group B (Targeted single biopsy by I‐scan OE); After careful endoscopic exam and identification by I‐scan OE, a single targeted biopsy was taken from either of the following gross or subtle abnormal mucosal patterns: (1) diffuse homogenous redness, (2) mucosal edema, (3) antral nodularity, (4) enlarged gastric fold, according to the following cascade: (1) overlapped abnormal mucosal patterns, (2) the most severe or prominent of nonoverlapped, coexistent patterns, (3) the most affected or severe part of a single pattern, (4) avoiding areas with suspected atrophic gastritis and IM with the aid of I‐san OE.2.1All procedures were performed with PENTAX Medical EPK‐i7010, i10, high definition (nonmagnifying) endoscopy with OPTIVISTA (I‐scan OE) technology, and by a single experienced endoscopist. Patients fasted for at least 6 h. The examination was done under either pharyngeal anesthesia (xylocaine spray), sedation (midazolam ± fentanyl), or both. The stomach's antrum, corpus, and fundus were carefully examined from a medium distance after good insufflations and adequate washing, initially by WLE, then followed by I‐scan OE moods which were turned on from 1 to 3 consecutively to assess the presence of the abnormal mucosal or vascular patterns or subtle abnormalities. Biopsies were then taken according to the study protocol with the same biopsy cup size. The followings are the abnormal mucosal patterns with endoscopic description featuresDiffuse homogenous redness: uniform redness in nonatrophic mucosa mainly located in the corpus . It should not be used to make treatment decisions.Its name originated from the Campylobacter‐like organism. CLOtest identifies the presence of urease enzymes, which hydrolyze urea into ammonia and bicarbonate, leading to a pH change indicated by a change in the color of phenol red from yellow (negative reading) to pink or red (positive reading). The biopsy specimens were inoculated into CLOtest kits at the same room temperature for 24 h with a reading time of the positive results at 1, 4, and 24 h. The CLOtest was discarded after 24 h  technology from PENTAX Medical, based on postprocessing of reflected light with the difference of reflective properties between the normal and abnormal mucosa.2.4t‐test was used for their analysis. Number and percentages were used for summarizing qualitative data, while the normal Z‐test was used to assess the significant difference between the two proportions. A p‐value of < 0.05 was considered statistically significant.The collected data were analyzed using SPSS (IBM SPSS Statistics 28.0). Mean, standard deviation (SD), and range was used for summarizing quantitative data, 3p = 0.98). The most common site for biopsy in group B (targeted) was the corpus (60.7%), followed by the antrum (28.6%), then the least was the fundus (10.7%). Table p = 0.501)]. However, 78.6% of the patients in group B had a positive CLOtest at 1 h vs. 60.7% in group A (p = 0.047), and 17.8% of patients in group A had a positive CLOtest at 24 h (late positive results) versus 5.3% in group B (p = 0.032). Therefore, the reading time needed for CLOtest to turn positive was shorter if the biopsy was targeted (not random) with the aid of I‐scan OE.Table e antrum 8.6%, the4H. pylori infection with high sensitivity; however, its false‐negative results are still apparent. Missing a site rich in H. pylori organisms could be because the biopsy is randomly taken or taken from a normal‐looking mucosa in the corpus or incisura as recommendedH. pylori load and activity.H. pylori load is present and overcome many causes of false‐negative results.H. pylori infection. The positive CLOtest is generally to be believed, however, a negative test requires histology or other confirmation tests with more cost and time, especially when H. pylori infection is likely the cause of the endoscopic finding, such as a bleeding peptic ulcer, or when a false‐negative result is suspected.H. pylori diagnostic work‐up.The CLOtest is a commonly used RUT for detecting H. pylori infection. There was no need for close observation in this study, which needs more examination time, as I‐scan OE technology could detect abnormal mucosal patterns at a medium distance from the gastric mucosal surface. On the other side, magnifying endoscopy (M‐Iscan) can reveal more precisely the subtle abnormal mucosal patterns in H. pylori infection; however, it requires more training, time, and experience,Cho et al.H. pylori organisms in one tissue sample, the patchy uneven distribution of the organism in the mucosa, or sampling of IM . Moreover, choosing UBT as the gold standard test to determine the sensitivity of the CLOtest, which is less likely to be susceptible to sampling error and may be more sensitive than histology. Thus, a biopsy‐based test, such as culture, histology, or CLOtest, may underestimate the presence of H. pylori in the stomach if the result is based on a single biopsy sample. In contrast, in this study, a single targeted biopsy with I‐scan OE technology produced high sensitivity and almost near to CLOtest sensitivity with four random biopsy samples taken from antrum in Siddique et al. and Lee et al., studies ,H. pylori infection. Biopsies for histology were taken in this study if suspected endoscopic mucosal lesions, such as IM or atrophy. Otherwise, the UBT was the used gold standard test, which was in agreement with other studies.H. pylori infection is time‐consuming, expensive, with a high risk for sample error, as previously mentioned.Usually, obtaining a single biopsy sample, especially from the antrum, for CLOtest is associated with a high sample error and very low test sensitivity. Siddique et al. reported a very low CLOtest sensitivity (52%) with a single random biopsy from the antrum and only 4% positive CLOtest readings after 1 h.In this study, the single targeted biopsy‐based CLOtest achieved a high sensitivity rate and overcame the previously mentioned sample error risk; however, there was no significant increase in the sensitivity over the nontargeted dual random biopsy‐based CLOtest (92.8% vs. 89.3%). On the other side, multiple biopsies for CLOtest have a higher risk of being missed, slipped, or crushed, especially if taken by biopsy forceps simultaneously or during incubation in the CLOtest kit, with resultant false‐negative results. The nontargeted dual biopsy in this study was taken separately and then combined outside before CLOtest incubation as recommended to increase sensitivity,H. pylori by UBT; it was not possible to calculate specificity for the two groups as there were no patients without H. pylori infection in both groups. Although H. pylori infection with normal‐looking endoscopy exam is present,H. pylori infection.The sensitivity of CLOtest with a single random biopsy is widely affected by the severity of gastritis with atrophy. Lan et al.5Subjective evaluation of the severity of mucosal abnormalities and preference of one over another in sampling and inter‐observational agreement is difficult to be standardized. It is limited to a single center. The nonmagnifying I‐scan technology, as magnifying I‐scan, could detect more subtle mucosal abnormalities, especially in apparently looking normal mucosa.6Sampling a targeted gastric biopsy with the aid of I‐scan OE technology for CLOtest significantly shortens the reading time of positive results with a high test sensitivity, allowing for early decision making and saving time.All the work is the sole author's (Hosam M. Dawod) effort.The author declares no conflict of interest.This study was obtained from the review board of Health Studies & Research Approval Committee (HSRAC), Centre of Studies & Research—Ministry Of Health, Sultanate of Oman. Informed consent was obtained from all patients recruited in the study.The sole author (Hosam Mohamed Dawod) affirms that this manuscript is an honest, accurate, and transparent account of the study being reported; that no important aspects of the study have been omitted; and that any discrepancies from the study as planned.


Honey has a very important commercial value for producers as a natural product. Honey aroma is formed from the contributions of several volatile compounds, which are influenced by nectar composition, botanical origins, and location. Selected‐ion flow‐tube mass spectrometry (SIFT‐MS) is a technique that quantifies volatile organic compounds simply and rapidly, even in low concentrations. In this study, the headspace concentration of eight monofloral  and three multiflower Turkish honeys were analyzed using SIFT‐MS. Soft independent modeling of class analogy (SIMCA) was used to differentiate honey samples based on their volatiles.This study focused on 78 volatile compounds, which were selected from previous studies of selected honeys. Very clear distinctions were observed between all honeys. Interclass distances greater than 8 indicate that honeys were significantly different. Methanol and ethanol were abundant in the honeys. Chestnut honey collected from the Yalova region had the highest total concentration of volatiles followed by heather honey and chestnut honey collected from the Düzce region.Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.Honeys with different botanical and geographical origins showed differences in their volatile profile based on chemometric analysis. Of the honey samples, methanol, ethanol, acetoin, ethyl acetate, and isobutanoic acid had the highest discriminating power. Methanol and ethanol, and then acetic acid, were the volatiles with the highest concentrations in most honeys. © 2020 The Authors.  Honey is one of the oldest foods in existence, and is consumed not only for its effects on health but also for its taste, nutritional value, and unique flavor. Honey contains mainly water and sugar.Honey has a very important commercial value for producers as a natural product.p‐anisaldehyde, and p‐cresol in heather honey.et al.,α‐pinene, β‐pinene, toluene and 1.2.3‐trimethylindene are marker compounds for pine honey. Characteristic volatiles of sage honey were tetrahydro‐2,2,5,5‐tetramethylfuran, lilac aldehyde, 2‐methylbenzene, heptanoic acid, and benzeneacetic acid.Volatile compounds present in honey are characteristic markers of botanical origin.The evaluation of the botanical and geographical origin of honey is very complicated. The fingerprint of specific honey samples can be determined by measuring organoleptic properties, melissopalynological characteristics, and physicochemical characteristics.The principal objective of this study was to determine if chestnut, rhododendron, lavender, sage, carob, heather, citrus, pine, and wildflower honeys can be distinguished based on their volatile organic compounds using a SIFT‐MS technique combined with multivariate statistical analysis.Twelve honey samples were selected from different parts of Turkey Table . Local bFor each honey sample, 10.02 ± 0.2 g was transferred into a 500 mL Pyrex bottle and capped with an open‐top cap, lined with a polytetrafluoroethylene (PTFE)‐faced silicone septa. The samples were kept in a temperature‐controlled water bath  at 50 °C for 60 min to allow equilibration of the volatiles, which were released from the honey samples into the headspace. Samples were measured in triplicate.3O+, NO+, and O2+. The concentration of the volatiles was measured by employing the predetermined reaction rate constant for the volatile with a selected precursor ion and accounting for the dilution of the sample gas into the carrier gas (helium) in the flow tube.3 min−1).A selected‐ion flow‐tube mass spectrometer  was used to measure and quantify the volatile compounds in the headspace. Selected‐ion flow‐tube mass spectrometry uses chemical ionization with selected positive reagent ions, H−1 in the headspace above the honey sample. During the analysis, some compounds produce the same mass for a given precursor ion, so the interfering compounds have to be reported as a mixture. In this study, several mixtures were identified at different charge‐to‐mass ratios, such as 2‐methyl‐2‐butanol and butanoic acid at 71 m/z, acetone and isoamyl alcohol at 88 m/z, acetic acid and 2‐cyclopenten‐1,4‐dione at 90 m/z, dimethyl disulfide and phenol at 94 m/z, acetic acid and p‐cresol at 108 m/z, acetoin and ethyl acetate at 118 m/z, phenylacetaldehyde and isopropyl benzene at 120 m/z, 2‐phenylethanol and santene at 122 m/z, dimethyl trisulfide and hydroxymethylfurfural at 126 m/z, α‐pinene, β‐pinene, 2‐hydroxyacetophenone and 4‐methoxybenzaldehyde at 136 m/z, octanoic acid and nonanol at 144 m/z, and hotrienol and p‐menth‐1‐en‐9‐al at 152 m/z.The range of the mass‐to‐charge ratio was set to 10–250 m/z, with a total SIM scan time of 120 s. The concentration of measured volatile compounds, which was calculated through known kinetic parameters, is listed in Table P ≤ 0.05 using SPSS . Multivariate statistical analysis was conducted using SIMCA with Pirouette software for Windows Comprehensive Chemometrics Modeling, version 4.0  to identify distributions of volatiles in honey samples.The concentrations of volatile compounds were analyzed in triplicate. One‐way analysis of variance (ANOVA) using Tukey's procedure with a 95% confidence interval was performed to determine statistical differences among samples; significance was defined as Many compounds have been detected in honey using different techniques. This study focused on 78 volatile compounds, which were selected from previous studies of selected honeys. Twelve honey samples of known botanical and geographical origins were analyzed Table . MethanoAcetic acid was the third highest concentrated volatile followed by methanol and ethanol in the honeys, except for chestnut honeys from Yalova region and lavender Table . Acetic Acetone was the fourth highest volatile of chestnut_Düzce, pine, wildflower_Ardahan and wildflower_Kırşehir, while 2‐cyclopenten‐1,4‐dione was the fourth leading volatile compound for rhododendron, heather and wildflower_Sivas. Alpha‐pinene was the fourth higest concentrated compound measured in sage honey, while ethyl acetate was measured in carob and dimethyl sulfide in citrus as fourth highest compound. Acetone is responsible for a pungent or fruity odor and ethyl acetate gives a fruity aroma in honey.Ethyl acetate, which is the ester formed from ethanol and acetic acid, was one of the most abundant compounds in carob honey after ethanol, methanol, and acetic acid. Some studies have focused on the volatile compounds found in carob;Persea americana (38.5%), Spartocytisus supranubius (25.2%), Quercus ilex (7.4–337%), Satureja montana (22.8%), and Agave honey (19.4%).Dimethyl sulfide was one of the compounds with the highest concentration detected in citrus after ethanol, methanol, and acetic acid. It was also found in both raw and heat‐treated citrus honey from Spain.Hotrienol is one of the marker volatiles for lavender honey;et al.Several alcohols were identified in lavender honey, and ethanol, methanol, isoamyl alcohol, lemonol, 2‐butanol, hotrienol and 1,3‐butanediol were the highest concentrations. Radovic et al.Multivariate statistical analyses allow the determination of botanical and geographical discrimination between honey samples. Interclass distances (ICDs) greater than 3 indicate that samples were significantly different.et al.Z)‐3‐hexen‐1‐ol, acetic acid, 2‐cyclopentene‐1‐4‐dione and butanoic acid were found to be higher in heather honey compared to citrus. These compounds were discriminating volatiles for heather and citrus. Even though it is difficult to determine the botanical source of honey accurately by many techniques,et al.When comparing different botanical sources from the same province, chestnut and rhododendron (Düzce), lavender and sage (Burdur), and carob, heather, and citrus  showed clear differentiation Fig. . The conp‐menth‐1‐en‐9‐al (Table et al. When the same flower source was compared with different locations, such as chestnut honeys from Yalova and Düzce, or wildflower honeys from three different provinces, varied volatile compositions were detected Fig. . The comal Table . ChestnuThe concentrations of 2,3‐butandione, 2‐butanol, 2‐cyclopenten‐1,4‐dione, acetic acid, acetoin, acetone, butanoic acid, ethyl acetate, furfural, isoamyl alcohol, isobutanoic acid, isopropyl benzene, maltol, methanol and phenylacetaldehyde were different in the three wildflower honeys from different locations. The aroma composition of wildflower honeys can be dissimilar from each other because of the variation and differences of flowers contingent upon the location.et al.Karabagias SIFT‐MS is a fast and simple method to enhance the difference between Turkish honeys based on their volatile composition. The application of SIFT‐MS technique with the aid of chemometrics for floral and geographical origin determination of honeys can be very useful. The data analysis takes place in two‐dimensional matrices with a chemometric approach, which allows for a better separation of the samples.Honeys with different botanical and geographical origin showed differences in their volatile profile based on their interclass distances. Between the honey samples, methanol, ethanol, acetoin, ethyl acetate, and isobutanoic acid had the highest discriminating power and also methanol and ethanol, and then acetic acid, were the volatiles at the highest concentration in most honeys. In general, chestnut from the Yalova region had the highest total concentration of volatiles followed by heather and chestnut from the Düzce region, and wildflower from the Ardahan region had the lowest total concentration.The volatile composition of each honey type was affected by several factors. Future studies with a broader variety of honeys or geographical origins with different harvesting seasons may be required for a better understanding of the honey fingerprint.


Meniscus tears are one of the most frequent orthopedic knee injuries, which are currently often treated performing meniscectomy. Clinical concerns comprise progressive degeneration of the meniscus tissue, a change in knee biomechanics, and an early onset of osteoarthritis. To overcome these problems, meniscal transplant surgery can be performed. However, adequate meniscal replacements remain to be a great challenge. In this research, we propose the use of a decellularized and sterilized human meniscus allograft as meniscal replacement.2). The decellularization efficiency of human meniscus tissue was evaluated via DNA quantification and Hematoxylin & Eosin (H&E) and DAPI staining. The mechanical properties of native, decellularized, and decellularized + sterilized meniscus tissue were evaluated, and its composition was determined via collagen and glycosaminoglycan (GAG) quantification, and a collagen and GAG stain. Additionally, cytocompatibility was determined in vitro.Human menisci were subjected to a decellularization protocol combined with sterilization using supercritical carbon dioxide (scCOHuman menisci were decellularized to DNA levels of ~ 20 ng/mg of tissue dry weight. The mechanical properties and composition of human meniscus were not significantly affected by decellularization and sterilization. Histologically, the decellularized and sterilized meniscus tissue had maintained its collagen and glycosaminoglycan structure and distribution. Besides, the processed tissues were not cytotoxic to seeded human dermal fibroblasts in vitro.Human meniscus tissue was successfully decellularized, while maintaining biomechanical, structural, and compositional properties, without signs of in vitro cytotoxicity. The ease at which human meniscus tissue can be efficiently decellularized, while maintaining its native properties, paves the way towards clinical use.The online version contains supplementary material available at 10.1186/s40634-022-00555-y. With over 850,000 surgical interventions each year in the United States alone, meniscus tears are among the most frequent orthopedic knee injuries . They arWhen a meniscus is  injured or degenerated beyond repair, a  meniscectomy is performed , which lThese complications can be diminished by using decellularized allografts. Decellularization is the process of removing allogeneic cellular components, thus reducing the risk of disease transmission, immune rejection and inflammation while maintaining the integrity of the extracellular matrix (ECM) , 32. For2) sterilization have been used to sterilize ovine menisci while preserving native tissue structure and mechanical properties  for 24 hours at 60 °C in an Eppendorf ThermoMixer® at 300 rpm.To measure DNA, GAGs, and hydroxyproline (HYP), samples were digested, according to Kim et al. . BrieflyN = 8), decellularized + sterilized (N = 8), and native (2x N = 8) samples using the Qubit dsDNA High Sensitivity assay (Thermo Fisher Scientific) according to the manufacturers’ protocol. Readings were taken using the Qubit® 2.0 Fluorometer and data was normalized to tissue dry weight, obtained before digestion.The double-stranded DNA (dsDNA) content was quantified in decellularized (N = 3), decellularized (N = 3), and decellularized + sterilized (N = 3) samples were embedded in OCT Compound (Tissue-Tek®) and frozen on dry ice. Embedded samples were sectioned longitudinally at 6 μm and were fixed in 3.7% formaldehyde (Merck) for 20 min.Tissue sections of native . Sections were incubated in Mayer’s Hematoxylin solution (Sigma-Aldrich) for 10 min, washed in distilled and tap water, incubated in aqueous Eosin Y solution (Sigma-Aldrich) for 1 min, and washed again. Finally, tissue sections were dehydrated in increasing series of ethanol and mounted with Entellan (Merck).To identify remaining cell nuclei after decellularization and sterilization, tissue sections were stained with 4′,6-diamidino-2-phenylindole dihydrochloride . Tissue sections were incubated in 1 μg/mL DAPI solution for 30 min and mounted with Mowiol (Merck).Mechanical properties of medial menisci samples of five donors were determined using an unconfined stress-relaxation ball indentation test. Samples were prepared according to a previously described protocol by Maier et al. . In shorFrom the data, relative compression, as an indicator for tissue viscosity, was calculated from the indenter position at the end of dynamic compression in the 5th test cycle. The Young’s modulus was determined during the 2nd step of each test cycle, calculated from the linear slope of the engineering stress-strain curve. And finally, the residual force, a measure of the tissue’s viscoelastic behavior, was defined as the recorded load at the end of the 3rd step (static compression) of each test cycle.N = 8), decellularized + sterilized (N = 8), and native (2x N = 8) meniscus was determined, as a measure of collagen, according to Huszar et al. [The total HYP content of decellularized . Hydrated tissue sections were incubated in Picro Sirius Red solution for 60 min and rinsed with 3% acetic acid and absolute ethanol. Then, tissue sections were dehydrated in absolute ethanol and mounted with Entellan.Alcian Blue was used to visualize the effect of decellularization and/or sterilization on glycosaminoglycans. In short, tissue sections were incubated in 3% acetic acid solution (Abcam) for 3 min, incubated in 1% Alcian Blue solution  dissolved in 3% Acetic Acid) for 30 min, and washed in 3% acetic acid, tap water, and distilled water. Subsequently, tissue sections were incubated in Nuclear Fast Red solution (Abcam) for 5 min, and washed again. Tissue sections were dehydrated in increasing series of ethanol and mounted with Entellan.N = 3), decellularized (N = 3), and decellularized + sterilized (N = 3) samples were embedded in OCT Compound and frozen on dry ice. Then, samples were longitudinally sectioned at 300 μm thickness. Meniscus sections were freeze-dried for 48 hours, punched to a diameter of 4 mm, in order to fit the well, and sterilized in serial dilutions of ethanol, 70%, 80%, 90%, and absolute, for 20 min each, followed by UV sterilization for 15 min. Once sterilized, the sections were washed in sterile PBS three times 20 min each and once in culture media  for 20 min. Finally, tissue sections were incubated in culture media overnight at 37 °C and 5% CO2.Tissue sections of native  release was performed using the CyQUANT Cytotoxicity Assay Kit (Invitrogen) according to manufacturer’s protocol. Experiments were performed on day 1, 4, and 7. Cells cultured on a flat polystyrene surface were used as a negative control (0% cytotoxicity) and the maximum LDH release as a positive control (100% cytotoxicity).Human dermal fibroblasts  at passage 7 were seeded on the meniscus tissue sections (2000 cells per tissue) and submerged in 100 μL culture media and incubated at 37 °C and 5% COp < 0.05. Results are expressed as mean ± standard deviation.Since there are structural differences between the medial and lateral meniscus, the meniscus from left or right knee, and within the meniscus itself , it was p < 0.001). The remaining DNA content in decellularized and sterilized meniscus tissue was 20.1 ± 4.2 ng DNA per mg tissue dry weight, also significantly lower than the native contralateral control, which contained 193.3 ± 63.8 ng DNA per mg tissue dry weight   were observed Fig. B, E, H. The viscoelastic behavior of meniscus tissue was assessed using an unconfined indentation stress relaxation test over several cycles. All samples were loaded to 7 N without signs of plastic deformation and the slopes of the load curves were found to be linear between 2 N and 5 N Fig. .p = 0.40) and 42 ± 4% , respectively)  Fig.A.Fig. 3Np > 0.99) and between the decellularized + sterilized and native counterparts , in each individual test cycle   Fig. C. For boTogether, these results indicate that the mechanical properties of native meniscal tissue are maintained after decellularization and/or sterilization.p = 0.88). Decellularized + sterilized samples were composed of 87.9 ± 7.7 μg hydroxyproline per mg tissue dry weight, which was not significantly different from native contralateral controls with 83.7 ± 7.2 μg/mg dry weight    and the LDH cytotoxicity was found to be equal to the spontaneous LDH release of the cells . Besides, the LDH release of human dermal fibroblast seeded on a standard flat polystyrene surface was not significantly different from that of the experimental groups . In addition, the LDH release was constant during 7 days of in vitro culture. The maximum LDH release increased over time , indicative for an increase in total cell number (proliferation).The LDH release of human dermal fibroblasts on human native, decellularized, and decellularized + sterilized meniscus tissue sections was quantified Fig. . There wCurrent strategies for meniscus replacement can be further optimized to provide a long-term chondroprotective effect and thus clinical outcome. For this reason, this study aimed to create a decellularized and sterilized human meniscus allograft while maintaining its mechanical, compositional, and structural properties. Results demonstrated that human meniscal tissue could be decellularized to meet previously set decellularization criteria . This also displays the robustness of our decellularization protocol, which was previously used to decellularize porcine Achilles’ tendon and porcine and human anterior cruciate ligament tissue . DecelluIn this research, decellularization and sterilization did not significantly affect biomechanical properties, suggesting that the mechanical behavior of human native meniscus tissue can be maintained. This is promising since previous research reported altered mechanical properties, e.g., a lower Young’s modulus and decreased hysteresis area and peak stress of hysteresis . MoreoveSince meniscal biomechanical properties are closely related to the structure and organization of the collagen network and GAG content, the maintained mechanical properties suggest that ECM components were not affected by decellularization and sterilization. Indeed, hydroxyproline content was not significantly altered upon decellularization and sterilization and no damaged collagen fibers were found histologically. This is in agreement with previous research into decellularized porcine and rabbit menisci , 34. SinMaintaining GAGs in decellularized tissue is important, but also experimentally challenging. GAGs increase water content and a loss in GAGs can decrease load-bearing capacity. In this research, no significant loss in GAGs was found upon decellularization and sterilization. Yusof et al.  and MoniDetergents have been used in several decellularization protocols and have been used to decellularize numerous tissues, varying from tendon, nerves, dermis, and trachea to heart valves . Two of In this study, in vitro characterization and validation of the decellularized and sterilized meniscus allograft have been performed. The decellularized human meniscus samples showed a dsDNA removal of around 90% and no visible cells and nuclear material on decellularized and sterilized H&E and DAPI stained tissue sections. Although it is claimed that tissues from which most of the visible cellular material is removed are safe to use for implantation , it is vThe major clinical challenge will be to have off-the-shelf meniscus grafts with a geometry that matches the (damaged) native meniscus, to fully restore its function. Proper load transmission is essential for achieving natural tibiofemoral contact pressures in absence of local stress concentrations, in particular at the native tissue – graft interface, which would increase the risk for re-ruptures . TherefoHuman meniscus tissue was successfully decellularized, while maintaining biomechanical, structural, and compositional properties. Moreover, processed human meniscus sections showed no signs of in vitro cytotoxicity. Together, these results reveal that a decellularized human meniscus allograft has excellent potential for the development of a tissue-engineered solution for off-the-shelf meniscus replacement.Additional file 1.


We conducted a single‐center, open‐label, dose escalation, and expansion phase I trial of the antiangiogenic multikinase inhibitor regorafenib in patients with advanced myeloid neoplasms. We enrolled 16 patients with relapsed/refractory acute myeloid leukemia (AML), myeloproliferative neoplasms (MPN), chronic myelomonocytic leukemia (CMML), or myelodysplastic syndrome (MDS). A 3 + 3 dose escalation design was used with two planned dose levels (120 or 160 mg daily) and one de‐escalation level (80 mg daily). An additional 10 patients were treated on an expansion cohort. The recommended phase two dose of regorafenib was 160 mg daily, with no dose‐limiting toxicities. The best overall disease response by International Working Group criteria included one partial and stable disease in 11 patients. Tissue studies indicated no change in Ras/mitogen‐activated protein kinase (MAPK) pathway activation in responders. Pharmacodynamic changes in plasma VEGF, PlGF, and sVEGFR2 were detected during treatment. Baseline proinflammatory and angiogenic cytokine levels were not associated with clinical response. Single‐agent regorafenib demonstrated an acceptable safety profile in relapsed/refractory myeloid malignancy patients. Most patients achieved stable disease, with modest improvements in cell counts in some MDS patients. Biomarker studies were consistent with on‐target effects of regorafenib on angiogenesis. Future studies should investigate the role of regorafenib in combination therapy approaches. The bone marrow microenvironment is an important regulator of hematopoiesis in hematologic malignancies. Malignant hematopoietic cells consume high levels of oxygen and secrete pro‐angiogenic molecules such as VEGF to support leukemic growth . MicroveVEGF inhibitors have been previously evaluated in myeloid malignancies given the essential role of this pathway in tumor angiogenesis. A phase 2 trial of bevacizumab, a recombinant monoclonal antibody targeting VEGF, with standard induction chemotherapy in newly diagnosed elderly AML patients showed no improvements in clinical outcomes . HoweverThese disappointing results may be related to the limitations of more selective angiogenesis inhibitors. A variety of mutations have been identified in the pathogenesis of myeloid malignancies, which result in activation of transcription or growth factor receptors such as but not limited to, RAS/RAF/Mitogen‐activated protein kinase/extra‐cellular signal regulated kinase (ERK) kinase (MEK)/ERK, Janus kinase (JAK)‐signal transducer and activator of transcription, and Fms‐like tyrosine kinase 3 (FLT3) pathways , 16, 17.22.1We conducted a single‐center, open‐label, dose escalation, and expansion phase I trial in patients with relapsed/refractory AML, MPN, CMML, or MDS to assess the safety, tolerability, and preliminary efficacy of regorafenib. A 3+3 dose escalation design was used with two planned dose levels (120 mg or 160 mg daily), as well as one de‐escalation level (80 mg daily) if needed. Three to six patients were evaluated per dose level, and dose escalation continued until the maximum tolerated dose (MTD) was identified, defined as the highest dose level where 0/3 or 1/6 patients experienced a dose‐limiting toxicity (DLT). For the purposes of dose escalation, a DLT was defined as grade ≥3 toxicity occurring within the first 28 days after initiation of treatment and unrelated to the underlying myeloid neoplasm. Toxicity was graded using Common Terminology Criteria for Adverse Events version 4.0. In the expansion phase of the trial, an additional 10 patients were enrolled at the MTD. The primary objective of this study was to assess the safety and tolerability of regorafenib in this patient population and to establish the recommended phase 2 dose (RP2D). The secondary objective was to assess the pharmacodynamics of regorafenib via changes in known regorafenib targets and to assess treatment response to regorafenib.Treatment consisted of regorafenib at the starting dose level of 120 mg daily for 3 weeks on and 1 week off and administered on repeating cycles of 28 days. Patients were followed and treated on an outpatient basis for physical examination, standard laboratory testing, and adverse event evaluation. All patients received a baseline bone marrow aspiration and biopsy within 28 days of enrollment and repeated at 1, 3, 6, and 12 months and if there was concern for disease progression or relapse. Correlative testing occurred at time points as described below. Patients were continued on regorafenib if they derived clinical benefit and were tolerating therapy. All patients who received a dose of regorafenib were included in analysis.The protocol was approved by the institutional review board and compliant with the Declaration of Helsinki and guidelines on Good Clinical Practice. All patients provided informed consent.2.2Eligible patients were adults (≥18 years) with a diagnosis of advanced myeloid malignancy, as defined by: relapsed/refractory AML with failure of at least one line of prior therapy; patients with MDS and CMML that have failed hypomethylating agents; or patients with MF or unclassified MPN on maximal tolerated ruxolitinib with persistent residual symptoms, splenomegaly, or inadequately controlled blood counts. Patients were required to have adequate organ function and Eastern Cooperative Oncology Group (ECOG) performance status of 0–2.Exclusion criteria included prior antineoplastic therapy in the last 14 days (except hydroxyurea); uncontrolled hypertension ; history of arterial or venous thromboembolism within the last 6 months of informed consent; coagulopathy or other bleeding diathesis; seizure disorder; active and significant heart disease, presence of nonhealing wound, ulcer, or fracture; interstitial lung disease; and pregnancy.2.3Exploratory analyses of biomarkers were evaluated in participants at baseline (C1D1 prior to first treatment dose) and on C1D8, C1D15, and C2D1. Whole blood was centrifuged and separated into plasma and cellular components. The following proangiogenic and proinflammatory biomarkers were performed on plasma with the Human Angiogenesis Panel 1 V‐PLEX kit  for VEGF, bFGF, sFLT‐1, PIGF, sTie‐2, VEGF‐C, and VEGF‐D; the Human Pro‐Inflammatory Panel 1 V‐PLEX  for IFN‐γ, IL‐10, IL‐12p70, IL‐13, IL‐1β, IL‐2, IL‐4, IL‐6, interleukin 8 (IL‐8), and TNF‐α; the V‐plex Human IP‐10 kit  for IP‐10 and VEGFR2 . All samples were run in duplicate in the CLIA certified core of the Steele Laboratories at Massachusetts General Hospital (MGH). The fraction of lymphocyte and myeloid subtypes from the total circulating mononuclear cells were counted by flow cytometry using the following markers: CD3, CD4, CD8, CD25, CD45, CD56, and CD127 (BD Biosciences). Protein extract was obtained from bone marrow aspirates and Peripheral blood mononuclear cells (PBMCs) from selected patients with mutations in MAPK pathway and analyzed by Western blotting for total and phosphorylated ERK.2.4The primary objective of this study was to evaluate the safety and tolerability of regorafenib in patients with advanced myeloid malignancies. All patients who received at least one dose of regorafenib were included for analysis. DLTs were assessed during cycle 1, and rates of toxicity were summarized descriptively.The secondary objectives of this study were to assess responses to regorafenib and evaluate potential pharmacodynamic biomarkers of regorafenib by measuring baseline and changes after treatment in known regorafenib targets. Disease response was defined by International Working Group (IWG) criteria for AML, MDS, and CMML patients respectively and IWG MPNs Research and Treatment (MRT) criteria for MF , 21, 22.For biomarker analysis, variables were summarized as median and interquartile ranges at each timepoint. Statistical analysis was performed using R 4.0. A Wilcoxon signed‐rank test was used to test the paired differences in biomarkers from C1D1 to C1D8. Plasma biomarkers were excluded if more than 50% of values were out of the range of detection. For the purposes of plasma biomarker testing, responders were defined as patients who obtained a partial response or had stable disease for at least 2 months. A Wilcoxon rank‐sum test was used to compare baseline biomarkers and changes in biomarkers from C1D1 to C1D8 between responders and nonresponders. A Bonferroni correction was used to adjust for multiple comparisons.33.1Six patients were enrolled during the dose escalation phase, three patients were enrolled at 120 mg daily, and an additional three were enrolled at 160 mg daily. There were no DLTs in either cohort. Therefore, the RP2D of regorafenib was identified as 160 mg daily. Ten additional patients were enrolled at this dose during the expansion phase.N = 5, 31%, 3 de novo and two transformed from antecedent MDS), MDS , CMML , MF , and MPN‐NOS . Eleven (69%) patients had one prior line of therapy, four patients (25%) had two prior lines of therapy, and one patient (6%) had ≥3 prior lines of therapy. Within the MDS patients, three patients (42.9%) were high or very‐high risk by international prognostic scoring system (IPSS) criteria, and both MF patients were intermediate‐2 or high‐risk by dynamic international prognostic scoring system (DIPSS) criteria.Patient characteristics are described in Table All patients have discontinued study therapy, including five (31%) due to progressive disease and two (12.5%) due to unacceptable toxicity. Dose modifications and delays occurred in four and two patients, respectively. The median duration of treatment was 52.5 days or 1.85 cycles (interquartile range (IQR): 19–100 days)RAS family, including two secondary AML patients with an HRAS and KRAS mutation, one AML patient with a KRAS mutation, one MDS patient with a KRAS mutation, and one MPN‐NOS patient with an NRAS mutation.All patients had next generation sequencing from bone marrow performed on 92 commonly mutated loci across 23 genes associated with hematologic malignancies, as described previously [3.2NRAS G12D mutation and experienced an increase in white blood cell (WBC) from 54 × 109/L to 92 × 109/L with 3.5% blasts within a week of starting regorafenib, which resolved after stopping the treatment. A follow‐up bone marrow biopsy showed no disease progression. Adverse effects are summarized in Table At the 120‐mg dose level, a single grade 3 event occurred and was an episode of preseptal cellulitis treated with intravenous antibiotics, possibly attributed to drug. There were no hematologic toxicities or liver function test liver function tests (LFT) abnormalities noted at the 120‐mg dose level. In the 160‐mg dose level and expansion cohort, approximately 20% of events were grade 3 or higher. The most common grade 3 or higher adverse effects were LFT abnormalities, fatigue, anemia, thrombocytopenia, and neutropenia, including two incidents of febrile neutropenia. Among the 16 patients enrolled, nine reported fatigue including three grade 3 AEs; eight patients had LFT abnormalities including three grade 3 AEs; and five patients reported anorexia (one grade 3 AE). Hematologic toxicities included anemia , neutropenia , and thrombocytopenia . Two patients discontinued regorafenib therapy due to unacceptable toxicity. No grade 3 or higher events occurred in cycle 1. One of these patients had a diagnosis of MPN‐NOS with an 3.3Figure We observed a modest improvement in cell counts in five patients with MDS and cytopenia, although this improvement did not meet IWG criteria for hematologic improvement given the short durations. Normalization of absolute neutrophil count occurred in all five patients, which was generally short‐lived. No improvements in hemoglobin or platelet parameters were seen.3.4n = 3) and RAS‐wild type disease (n = 2). We found high levels of ERK activation in all samples, which were maintained after regorafenib treatment irrespective of RAS‐mutation status  using bone marrow aspirates and PBMCs collected before and during regorafenib treatment from patients with RAS‐mutant disease (ease n = . We founp = 0.04) and PIGF , and a significant decrease in sVEGFR2 . Increases in plasma sTIE‐2 and VEGF‐D were not significant when corrected for multiple comparisons. Pro‐inflammatory biomarkers that showed significantly increased concentration from C1D1 to C1D8 included TNFα . There were also increases in plasma IL‐8, IL‐6, and IFN‐γ but these changes were not statistically significant when adjusted for multiple comparisons.We also examined the changes in soluble blood biomarkers Figure . From C1p = 0.03), although this did not retain significance when corrected for multiple comparisons.There were no significant associations between biomarkers at C1D1 and clinical response. An increase in plasma IL‐8 from C1D1 to C1D8 was associated with improved regorafenib response , although this was not significant when corrected for multiple comparisons. We detected no other significant changes in the fractions of circulating lymphocyte populations.Finally, we examined the changes in cellular blood biomarkers after regorafenib treatment Table . We foun4We report the first clinical trial to evaluate feasibility and biomarkers of regorafenib in patients with advanced myeloid malignancies. Regorafenib is an oral multikinase inhibitor that is food and drug administration (FDA) approved for treatment of multiple solid tumor types and has broad inhibitory activity of multiple kinases involved in proliferation, tumor immunity, and the tumor microenvironment. This makes regorafenib an appealing drug candidate in advanced myeloid malignancies, which is characterized by abnormalities in multiple signaling pathways.We found that regorafenib was well tolerated at a starting dose of 160 mg daily. Phase I data showed no DLTs and mostly grade 1/2 AEs. LFT abnormalities, fatigue and anorexia occurred frequently in our study, consistent with reported adverse events with regorafenib in other tumor types , 25, 26.NRAS‐mutated MPN patient experienced hyperleukocytosis with an accompanying increase in peripheral blasts within 1 week of starting regorafenib. This normalized after discontinuing therapy, and follow‐up bone marrow biopsy showed no evidence of disease progression. In addition to anti‐angiogenic effects, regorafenib has broad inhibitory activity in the RAS/RAF/MEK/ERK (MAPK) signaling pathway. Paradoxical activation of the MAPK pathway with RAF inhibition, as evidenced by ERK activation, has been documented in the presence of oncogenic RAS mutations, including worsening of an occult Ras‐mutated CMML in a melanoma patient treated with vemurafenib [RAS mutations, experienced signs of leukemic disease proliferation, and one patient with RAS‐mutated MDS remained on regorafenib for over a year with stable disease. We also did not see any signals for an association between RAS mutation status and disease response or AEs. However, further evaluation of regorafenib in RAS‐mutated leukemias should include close hematologic monitoring for signs of disease acceleration caused by potential paradoxical activation of ERK signaling upon RAF inhibition. Interestingly, we found no inhibition of MAPK pathway in samples from three RAS mutant or two RAS wild‐type cases. Moreover, of these three patients, one had prolonged stable disease on regorafenib for over 1 year, and one patient had initial partial response, suggesting that MAPK activation may not be the mechanism of benefit or indicate regorafenib treatment failure.Interestingly, one urafenib , 28. HowThe results of the pharmacodynamic biomarker studies were consistent with regorafenib's known anti‐angiogenic activity. We did not find associations between baseline angiogenic or proinflammatory cytokines with clinical outcomes when correcting for multiple comparisons. However, we found an increase in IL‐8 was associated with clinical response. IL‐8 is an inflammatory chemokine that has known associations with disease severity and overall survival in advanced myeloid malignancies such as MF . Its sigAlthough the primary objective of our study was safety, we found evidence for limited clinical activity of regorafenib in this advanced population as most patients experienced stable disease. The median duration of treatment was relatively short at approximately two cycles, with some lower‐risk MDS patients showing more durable responses, including one for over 1 year. These responses are consistent with regorafenib's overall benefits of disease stabilization. Studies of regorafenib in hepatocellular carcinoma for instance have noted overall survival (OS) improvement with few complete responses, including reduction of arterial enhancement and increased central tumor necrosis without changes in tumor volume , 30. GreIn conclusion, phase I data showed that regorafenib has an acceptable safety profile in relapsed/refractory myeloid malignancy patients, a population where few treatment options exist. Regorafenib induced stable disease in most patients and modest improvements in cell counts in MDS patients. Future studies should investigate which leukemic subpopulations may derive benefit from regorafenib and evaluate the potential role of regorafenib in combination therapy.Amir T. Fathi: Consulting: Foghorn, Amgen, Celgene, BMS, Agios, Novartis, Ipsen, Forma, Abbvie, Genentech, Morphosys, and Takeda. Donna Neuberg: Stock ownership in Madrigal Pharmaceuticals, and received research funding from Pharmacyclics. Yi‐Bin Chen: Consulting: Daiichi, Abbvie, Equilium, Incyte, Jasper, Celularity, and Actinium; clinical trial support: Abbvie, Celgene, BMS, Agios, and Servie; Dan G. Duda received consultant fees from Simcere, Surface Oncology, Sophia Bioscience, Innocoll and BMS, and research grants from Exelixis, BMS, and Surface Oncology. Dan G. Duda's research is supported by National Institutes of Health (NIH)  and Department of Defense PRCRP (grant numbers: #W81XWH‐19‐1‐0284 and W81XWH‐21‐1‐0738). Gabriela Hobbs: Consulting fees from Abbvie, Incyte, Blueprint, Novartis, and Keros; research support from Bayer, Incyte and Constellation; grants: ECOR PSDA Award and ASH‐AMFDP award. The other authors declare no potential conflict of interest. Result of this study has previously been reported as an abstract at the 2020 American Society of Hematology Annual Meeting . The funders had no role in data analysis or the preparation of this article.Joan How wrote the manuscript. Siyang Ren analyzed and interpreted data and performed statistical analysis. Jennifer Lombardi‐Story, Meghan Bergeron, Julia Foster, Phillip C. Amrein, Andrew M. Brunner, Amir T. Fathi, Hanno Hock, Jenna Moran, Rupa Narayan, Aura Ramos, Tina Som, and Meghan Vartanian performed research and collected data. Joan How, Anna Khachatryan, Hiroto Kikuchi, and Mei Rosa Ng performed research, collected, analyzed, and interpreted data. Yi‐Bin Chen, Dan G. Duda, and Gabriela Hobbs designed and performed research, collected, analyzed, and interpreted data.All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation  and with the Helsinki Declaration of 1975.


Ursus maritimus are threatened by sea‐ice loss and represent a subsistence resource for Indigenous people. We applied a novel population modeling‐management framework that is based on species life history and accounts for habitat loss to evaluate subsistence harvest for the Chukchi Sea (CS) polar bear subpopulation. Harvest strategies followed a state‐dependent approach under which new data were used to update the harvest on a predetermined management interval. We found that a harvest strategy with a starting total harvest rate of 2.7% (˜85 bears/yr at current abundance), a 2:1 male‐to‐female ratio, and a 10‐yr management interval would likely maintain subpopulation abundance above maximum net productivity level for the next 35 yr (approximately three polar bear generations), our primary criterion for sustainability. Plausible bounds on starting total harvest rate were 1.7–3.9%, where the range reflects uncertainty due to sampling variation, environmental variation, model selection, and differing levels of risk tolerance. The risk of undesired demographic outcomes  was positively related to harvest rate, management interval, and projected declines in environmental carrying capacity; and negatively related to precision in population data. Results reflect several lines of evidence that the CS subpopulation has been productive in recent years, although it is uncertain how long this will last as sea‐ice loss continues. Our methods provide a template for balancing trade‐offs among protection, use, research investment, and other factors. Demographic risk assessment and state‐dependent management will become increasingly important for harvested species, like polar bears, that exhibit spatiotemporal variation in their response to climate change.Climate change threatens global biodiversity. Many species vulnerable to climate change are important to humans for nutritional, cultural, and economic reasons. Polar bears  Climate change is projected to result in loss of species  polar bear subpopulation ranges across the northern Bering, Chukchi, and East Siberian seas , and practical aspects of harvest that managers can control, including the harvest rate and sex ratio, management interval, and level of precision in population data. In this paper, “harvest” refers to all human‐caused removals . With estimates of vital rates and abundance from an integrated population model for CS bears  were not included as individuals in the life cycle, but rather were used to define the reproductive status of their mother. Following Regehr et al. (N] to carrying capacity [K]) at a rate inversely proportional to its elasticity, according to density‐dependent curves developed previously for polar bears  was calculated as the log of λ, the dominant eigenvalue (Caswell R) defined as λ − l , the subpopulation size that results in the greatest net annual increment in numbers resulting from reproduction minus losses due to natural mortality, N~t is an estimate of abundance in year t from a research study, selected as the 15th percentile of its sampling distribution to protect against overharvest when sampling uncertainty is large  is the male harvest level, and SR is a factor that specifies the male‐to‐female ratio in removals. For context, we note that user‐specified values of F0 = 1 and SR = 1 would result in a total harvest level  equal to the theoretical maximum sustainable yield, provided that subpopulation size was equal to MNPL, the parameters R~MNPL and N~ were set at the true values of RMNPL and N, and the subpopulation consisted of 50% females.During population projections, harvest at each time step was calculated as follows:ach Wade ), 0.5 isht=Hfemalet+Hmalet/N~t. During simulations, we imposed an upper limit of 0.10 on h to mitigate demographic risk associated with large uncertainty in estimates of rMNPL from the CS‐IPM  removed annually. We evaluated the effects of this constraint by performing supplemental simulations with a higher upper limit on htotal , and uncertainty (rsd.mod). The mg.int specified the number of years between changes to the harvest based on new population data. During population projections, N~t=1 and R~MNPLt=1 were obtained from the CS‐IPM. On subsequent time steps, we simulated research studies that provided new data to inform harvest management by selecting values of R~MNPLt and N~t from a multivariate normal distribution, with means corresponding to true values in the projection and a covariance structure calculated from the CS‐IPM. This approach assumed stationary statistical relationships among parameters. Uncertainty in simulated research studies was calculated as the product of a modifier (rsd.mod) and the relative standard deviation (rsd) of estimates of RMNPL and N from the CS‐IPM (see We explored a series of harvest strategies defined by unique combinations of F0 (range 0–2) and SR (range 1–2) in Eqs. Sets A, B, and C of simulations included 72, 90, and 42 simulations, respectively. Each simulation consisted of ˜10,000 stochastic projections using the same starting vital rates  density effects were imposed through the density‐dependent curves of the vital rates and the submodel for Allee effects. Every several years, as specified by mg.int, simulated research studies provided new values of R~MNPLt and N~t that were used to calculate harvest until the next management interval. To incorporate sampling uncertainty, the matrix model was parameterized at t = 1 with estimates of vital rates  and abundance from one iteration of the Markov chain Monte Carlo results from the CS‐IPM. To incorporate environmental variation, five projections were performed for each vital rate sample while allowing for stochastic variation in projected K .K2: The linear model was fit to the number of ice‐covered days during 2000–2016, resulting in faster declines  and more interannual variation. K2 represents the assumption that future sea‐ice declines will occur at the rate observed since a physical regime shift in the CS region in the year 2000  and data precision (rsd.mod) were fixed to 10 yr and 1.0, respectively. The harvest sex ratio (SR) was 2.0  and three precision levels . For example, rsd.mod = 0.50 corresponded to future estimates of demographic parameters with relative standard deviations half as large as the CS‐IPM. For set B, carrying capacity followed K1 and SR was fixed to 2.0 . Carrying capacity followed r = 0.05. We included scenario 2 because survival estimates for independent bears from the CS‐IPM may have included negative bias due to un‐modeled heterogeneity in recapture and movement probabilities  <0 or >0.10. This resulted in removing 29% and 28% of samples from scenarios 1 and 2, respectively  was estimated as the proportion of stochastic projections for which Nt=36>0.70K. We also recorded median annual harvest at time steps t = 1, 18, and 36 (Ht=11836); median long‐term yield, defined as the 50th percentile of the average annual harvest over the 35‐yr duration of projections (H¯yield); probability of functional extirpation (Pext), defined as N < a quasi‐extinction threshold of 100 independent bears; and probability of male depletion (Pdep) due to sex‐selective harvest, defined as <50 males in life cycle stage 10.Our primary criterion for sustainability was to maintain subpopulation abundance above MNPL, which protects against overharvest while allowing the possibility for harvest levels to approach maximum sustainable yield USFWS . To estaPMO = 0.90 would have a 10% probability  of failing to meet the management objective, and thus would be considered sustainable at low risk tolerance. These levels of risk tolerances are used to summarize results and are not intended to represent the perspectives of an individual or organization. When a simulation set included harvest strategies that were incrementally higher and lower than would meet the management objective at a predetermined degree of risk tolerance, we used linear interpolation to approximate the specific strategy that would meet the objective.We report results for the subset of harvest strategies corresponding to three predetermined degrees of risk tolerance. We defined low, medium, and high risk tolerances as accepting a 10%, 30%, or 50% probability of failing to meet the management objective, respectively. For example, a harvest strategy with htotal = 2.0%  independent bears, calculated as the product of the distribution of total abundance and the stable‐stage proportion of independent bears . Under vital rate scenario 1 (Appendix rMNPL = 0.02 (95% CRI = 0.00–0.06). Density dependence functioned like Regehr et al. (N/K = 0.73 (95% CRI = 0.69–0.74) and a ratio of subpopulation growth rate at MNPL to maximum intrinsic growth rate  of 0.84 (95% CRI = 0.82–0.84). Under vital rate scenario 2 (Appendix rMNPL = 0.04 (95% CRI = 0.00–0.07). MNPL occurred at N/K = 0.70 (95% CRI = 0.68–0.74) and the ratio rMNPL/rmax was 0.83 (95% CRI = 0.82–0.84).During population projections, abundance at r et al. , with MNK required reductions in the harvest level to limit the risk of undesired demographic outcomes. Under vital rate scenario 1 (lower population growth rate) and across the three assumptions for future changes in K, harvest strategies that met our management objective of maintaining abundance above MNPL ranged from a starting harvest level Ht=1 = 33 bears/yr at low risk tolerance to Ht=1 = 85bears/yr at high risk tolerance  and across the three assumptions for future changes in mg.int and rsd.mod) led to increased sustainable harvest and long‐term yield. We present results from set B of simulations as proportional changes in sustainable harvest under different combinations of mg.int and rsd.mod, relative to a baseline harvest strategy with mg.int = 10 yr and rsd.mod = 1.0  were achieved by reducing mg.int from 10 to 5 yr or by increasing the precision of population data by changing rsd.mod from 1.0 to 0.25. Combinations of more frequent and precise population data outperformed changes to only one factor.More frequent and precise population data .Increasing the male‐to‐female sex ratio in the harvest (SR) led to moderate increases in sustainable harvest. For example, under scenario 1 at medium risk tolerance, 1htotal, led to moderately higher demographic risk when vital rate estimates were imprecise . Otherwise, patterns in results were similar  and faster (K2) declines in future carrying capacity corresponded to reductions in K of 3% and 9% per decade, respectively. Our findings demonstrate that, under state‐dependent management, a primary effect of declining K is that harvest levels must decline to avoid heightened demographic risk. Although harvest levels declined in our simulations, habitat loss did not require commensurate reductions in the harvest rate because compensatory effects allowed removal of some animals that, in the absence of harvest, would have died from crowding and competition.An advantage of our framework compared to earlier harvest risk‐assessment methods for polar bears  led to a 9% increase in long‐term yield  because such details are difficult to predict and management decisions for the CS subpopulation are made annually . For example, under vital rate scenario 2, reducing the relative standard deviation of estimated parameters by 50%  side of the harvest yield curve USFWS . BecauseHt=1 = 86 bears/yr  and the state‐dependent framework would provide opportunities to adjust the harvest, if necessary.Our estimates of harvest risk can be approached by comparing demographic outcomes for a moderate harvest strategy, under both vital rate scenarios, and then evaluating pros and cons of lower or higher harvests. Under vital rate scenario 2, a low risk‐tolerance strategy had a starting harvest level N, and periodically updating the harvest rate and level using new population data, is more robust than a fixed‐rate or, especially, a fixed‐level harvest strategy , when considering harvest strategies that met the management objective at medium risk tolerance , increasing the upper limit on htotal led to average reductions of only 4% in both Ht=1 and H¯yield , or negative density‐dependent effects that are more severe than our analyses, would lead to higher probabilities of undesired outcomes, including extirpation. A conservative approach to harvest management is one way to reduce demographic risk. Other options include ongoing monitoring of environmental, ecological, or demographic indices that can trigger changes to research or management , or establishing a multi‐level system under which graduated management and conservation actions are tied to pre‐established population thresholds . Our findings should be interpreted with caution given that continued warming could result in complex ecological interactions (e.g., between prey status and habitat availability; Stirling Appendix S1Click here for additional data file.Appendix S2Click here for additional data file.Appendix S3Click here for additional data file.Appendix S4Click here for additional data file.Appendix S5Click here for additional data file.Appendix S6Click here for additional data file.


Hybrid and vaccine‐induced immunity against SARS‐CoV‐2 in patients with MS. Ann. Clin. Transl. Neurol. 2022;9(10):1643‐1659. In the originally published version of the article, the co‐author's name was erroneously rendered as Igda Stanzin and has now been corrected to Idga Stanzin.We apologize for this error.