In early 2020, a novel coronavirus leading to potentially death was discovered. Since then, the 2019 coronavirus disease (COVID-19) has spread to become a worldwide pandemic. Beyond the risks strictly related to the infection, concerns have been expressed for the endocrinological impact that COVID-19 may have, especially in vulnerable individuals with pre-existing endocrinological health conditions. To date new information is emerging regarding severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) in children but the literature is still scarce concerning this infection in patients with intracranial malignant neoplasms. We report a 9-year-old child infected with SARS-CoV-2 and recent diagnosis of suprasellar non-germinomatous germ cell tumor also suffering from diabetes insipidus and hypothalamic-pituitary failure  and its clinical course. The patient remained asymptomatic for the duration of the infection without requiring any change in the replacement therapeutic dosages taken before the infection. We then discuss the proposed approach to treat a pediatric patient with SARS-CoV-2 infection and hypothalamic-pituitary failure and we include a review of the literature. Our report suggests that SARS-CoV-2 infection is usually mild and self-limiting in children even those immunocompromised and with multiple endocrinological deficits. Patients are advised to keep any scheduled appointments unless informed otherwise. The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which first appeared in December 2019 in Wuhan, has caused the coronavirus disease 2019 (COVID-19) related pandemic and has been declared a significant threat to international health by the World Health Organization.Based on data from large studies, obesity emerged as a strong and independent determinant of increased risk of morbidity and mortality in adult patients infected with SARS- CoV-2  and patiManagement of patients with pituitary tumors and their associated hormonal abnormalities and mass effect complications can be challenging even during normal times and need to be preferably handled in Pituitary Centers of Excellence, with a multidisciplinary approach, especially in complex cases. Some of the diagnostic and management dilemmas regarding adult patients with pituitary tumors, and guidance on safe and as effective as possible delivery of care in the COVID-19 era, are have recently been reported . Indeed,We report the case of a 9-year-old Caucasian female affected by suprasellar non-germinomatous germ cell tumor (NGGCT) associated with multiple pituitary deficit, who tested positive for SARS-CoV-2 an April 1, 2020.Her problem began 2 years earlier, with polyuria, polydipsia  and growth delay. In October 2019, she came to Verona’s Paediatric Emergency Department in a state of confusion. Brain magnetic resonance imaging revealed a suprasellar mass, compatible with germinoma Figure 1In October 2019 chemotherapy according to SIOP CNS GCT II standard risk protocol for non-germinomatous germ cell tumor (NGGCT) was started. The first cycle was complicated by hydrocephalus that required ventricular-peritoneal derivation. At the end of the third cycle, the MRI showed a partial response to chemotherapy. Therefore, in January 2020, the patient underwent neurosurgery. The histopathological examination was conclusive for immature teratoma. Follow-up MRI one month after surgery showed a suspected small residual lesion. Chemotherapy with high-dose etoposide was started.2.day divided into 2 doses. Therapy with recombinant GH was not yet started. Since her neurosurgical operation, she suffered from hypothalamic obesity  with massive weight gain.From the 8 March, the “lockdown” for all Italy was adopted together with the use of surgical mask, hand hygiene, and social distancing, to prevent the massive spread of SARS-CoV-2; moreover, a screening program by nasopharyngeal swab (NPS) for any patient and caregiver parent accessing the Pediatric Hematology Oncology Unit was started . On Marc2 .On March 5 she was admitted to the Pediatric Oncology Unit for mobilization chemotherapy with high-dose etoposide and granulocyte colony-stimulating factor (G-CSF) followed by peripheral stem cell collection for autologous stem-cell transplantation (ASCT). On April 1 she was admitted to the Pediatric Oncology Unit for ASCT. Her weight was 40,9 kg  and her height was 120,2 cm , with a BMI of 28,31 kg/m6/kg CD34+. The patient engrafted on day + 9 for neutrophil and on day + 12 for platelets and was discharge home in good clinical conditions. In accordance with Verona University and University of Verona Institutional review Board (IRB) policies, case reports are not considered to be research subject to IRB review and are thus except from formal IRB approval for publication. Written informed consent was obtained from the patient’s legal guardians for the publication of any potentially identifiable images or data included in this article.When she came for screening, a second NPS for SARS-CoV-2 was performed on the patient and parent caregiver (the father) and both resulted positive. The pre-transplant conditioning regimen  was already started on April 2 in the morning, and was therefore withdrawn as soon as the result of NPS was communicated in the evening of the same day (administration of day 1 of conditioning regimen). Both patient and father were quarantined in a pediatric COVID19 section of the department to monitor the patient’s symptoms or signs of SARS-CoV-2 infection. During observation, blood count, urine tests. inflammatory markers, chest X-ray were unremarkable. In particular, her blood exams showed leukocytes 6,87x10^9/L, neutrophils 5,77x10^9/L and lymphocytes 0,40x10^9/L. No respiratory, cutaneous signs, or fever appeared during recovery and the patient continued her previous therapies. The most significant laboratory tests before, during and after Sars-Cov2 infection are summarized in Our case report, for the first time, describes the evolution of SARS-CoV-2 infection in a child with intracranial malignant neoplasms and multiple pituitary failure. Clinical features of SARS-CoV-2 infection in adults have been reported elsewhere. Compared to adult patients, reported cases of pediatric patients had clinically milder symptoms and showed fewer alterations in radiological and laboratory testing parameters. A systematic review confirmed that Chinese pediatric patients with COVID-19 had a good prognosis and recovered within 1 to 2 weeks after disease onset, and no cases of pediatric death from COVID-19 were reported in the age range of 0 to 9 years. One death was reported in the 10 to 19 years range, but no further information was provided on this patient .Primary central nervous system (CNS) germ cell tumors (GCT) are a heterogeneous group of tumors that are still poorly understood. Most of these tumors develop along the midline, mainly from the pineal gland, followed by tumors arising in the suprasellar cistern . The supOur patient was affected by non-germinomatous germ cell tumor (NGGCT), based on alfa-fetoprotein, hCG levels in plasma and CSF and based on histopathological examination. The girl had secondary adrenal insufficiency, diabetes insipidus, central hypothyroidism, hypothalamic obesity and GH deficiency.How to manage patients with pituitary tumors without easy access to full investigations in the COVID-19 era was described by Fleseriu et al.  and the Our patient had long suspended the high antiedema doses of dexamethasone at the time of infection and she was taking replacement doses of hydrocortisone. We presumed that she was infected through contact with her father at home, and she tested positive regardless of the therapies she was taking.The impact of COVID-19 on patients with pituitary-neuroendocrine disease also needs to be considered. Included in the management of a patient with hypopituitarism are replacement of hormone deficiencies, regular screening for the development of new pituitary hormone deficiencies, surveillance of the underlying cause . Many of these patients have hypopituitarism including secondary adrenal insufficiency, requiring stress dose glucocorticoid supplementation in adult patients and in children . Also, pOur young patient never showed signs or symptoms indicating clinical deterioration, which in patients affected by COVID-19, typically occur 7-10 days after the onset of the first COVID-associated symptoms. Our report confirms that an asymptomatic child, who tested SARS-CoV-2 positive, for example through family or oncology screening, does not need to increase their routine hydrocortisone replacement dose. However, we stress that, in case of an acute COVID-19 infection with clinical deterioration, children with adrenal insufficiency should immediately receive a parenteral injection of 50-100 mg/m2 hydrocortisone  .No published reports so far have reported a higher prevalence of dysnatremia in COVID-19. However, the COVID-19 pandemic has limited the accessibility of blood testing, and the priority of routine treatment of central DI should be to avoid hyponatremia. It emphasizes the importance of delaying desmopressin doses, to allow regular periods of free water clearance, so that excess water intake does not lead to dilutional hyponatremia . Our patData on thyroid function or thyroid pathology are not yet available in COVID-19. Patients with underlying hypothyroidism or hyperthyroidism are advised to continue their prescribed medications as usual . Our patFinally, the first systematic review dealing with the impact of immunosuppression on SARS-CoV-2 infection affirmed that immunosuppressed hosts may not present a greater risk of an increased severity of disease, compared to the general population . ConsideDuring this COVID-19 pandemic, in children with hypopituitarism, proper methods for viral respiratory infection control and prevention, supportive therapies, and considerations of potential therapies relevant to the pituitary disorders and any underlying comorbidities should be undertaken. The caregivers are urged to request for COVID-19 testing for patients if exposed. Even if symptoms are mild or if fever is present contacting the team  is mandatory in order to seek medical guidance. Maintain of adequate hydration, adhere to daily medications, and adapt to increased replacement dosages if clinically indicated, is recommended.Recognizing SARS-CoV-2 as a possible causal trigger of endocrinological failure and its exacerbation is of particular importance to improving its early diagnosis. In addition, the aim of our report is to be informative while reassuring at the same time. Our case suggests that SARS-CoV-2-infection is usually mild and self-limiting in children even in those who are immunocompromised. Patients are advised to keep any scheduled appointments unless informed otherwise.The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.RG designed the study. VO, MC, and PC collected data. RG and PC performed data interpretation. RG and VO wrote the first draft of the manuscript. RG, SC, AF, and GP critically reviewed the manuscript for intellectual content. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


A very thin polymer film of about 110 nm is achieved with the spin-coating technique. The thickness of the gain medium can support only the zero-order transverse electric lasing mode. We expect that such a low threshold lasing device can find application in optoelectronic devices.In recent years, conjugated polymers have become the materials of choice to fabricate optoelectronic devices, owing to their properties of high absorbance, high quantum efficiency, and wide luminescence tuning ranges. The efficient feedback mechanism in the concentric ring resonator and its circularly symmetric periodic geometry combined with the broadband photoluminescence spectrum of the conjugated polymer can generate a highly coherent output beam. Here, the detailed design of the ultralow-threshold single-mode circular distributed feedback polymer laser is presented with combined fabrication processes such as electron beam lithography and the spin-coating technique. We observe from the extinction spectra of the circular gratings that the transverse electric mode shows no change with the increase of incident beam angle. The strong enhancement of the conjugated polymer photoluminescence spectra with the circular periodic resonator can reduce the lasing threshold about 19 µJ/cm In optoelectronic and also in laser technology, gain materials are regarded as the building block that can improve device performance, and some of them open a new route towards real-life applications. Similarly, common gain materials, such as inorganic semiconductors, organic dyes, organic semiconductors, quantum dots, and perovskites, are used in laser systems with small differences in thin film fabrication properties, electrical conductivity, and difficulties in the manufacturing process ,6,7,8,9.Recently, various strategies have been proposed to engineer the operating characteristic of the second-order and high-order DFB polymer lasers, especially to control the output-coupled lasing beam (surface-emitting) with low divergence angle along and normal to the periodic nanostructures ,28,29. Tn (integer for grating order), respectively. Therefore, surface-emitting lasing from the circular grating structure is obtained via first-order diffraction (β = 0) and the above equation can be further modified as; In the above equation, In this article, we focus on the morphology and lasing performance of optically pumped surface-emitting circular DFB polymer laser. The solution of PMMA in chlorobenzene was spin-coated on a glass (BK7) substrate, and after the baking process circular grating structures were written with electron beam lithography (EBL). The gain material conjugated polymer F8BT was spin-coated on the prepared circular grating structures. Finally, the circular DFB polymer laser was optically pumped to investigate the lasing properties such as lasing thresholds and emission wavelength. In this work, we applied electron beam lithography, a simple and effective fabrication technique for writing the geometry of circular periodic structures on the glass substrate, which would provide essential feedback mechanism for attaining the surface-emitting circular DFB polymer laser. Improved insight into the developed circular grating DFB polymer laser system can be gained by investigating its various properties such as morphology of the circular grating nanostructures, optical microscopic images, optical spectroscopic characterization, and lasing properties. The morphology of the circular grating structure was studied through the scanning electron microscope (SEM) to check the quality of the feedback cavity. The SEM photographs of the circular periodic nanostructures on the glass substrate at various magnification powers are depicted in the In our experiment, we spin-coated a thin film of F8BT on the circular and regular (1D) grating structures to study the angle resolved polarization properties of the TE mode. Both the samples were excited with a non-polarized white light from tungsten halogen lamp . The optical transmission spectra were measured by using fiber spectrum analyzer from optical spectrometer . The incident white light can be TE polarized, when its electric field is parallel to the incident grating planes. Extinction spectrum for both the circular and regular gratings  covered with a gain material F8BT can be achieved from the formula In our experiment, we studied the single as well as quations :(2)de= technique was applied to realize a very thin film of about 110 nm from the gain material F8BT. The optical extinction spectra section in our experiment can provide sufficient information about the efficient enhancement of the circular periodic geometry with the photoluminescence spectrum of the conjugated polymer F8BT. As a result, we have realized low-threshold (approximately


The evolution of SARS-CoV-2 pneumonia to acute respiratory distress syndrome is linked to a virus-induced “cytokine storm”, associated with systemic inflammation, coagulopathies, endothelial damage, thrombo-inflammation, immune system deregulation and disruption of angiotensin converting enzyme signaling pathways. To date, the most promising therapeutic approaches in COVID-19 pandemic are linked to the development of vaccines. However, the fight against COVID-19 pandemic in the short and mid-term cannot only rely on vaccines strategies, in particular given the growing proportion of more contagious and more lethal variants among exposed population . As long as collective immunity is still not acquired, some patients will have severe forms of the disease. Therapeutic perspectives also rely on the implementation of strategies for the prevention of secondary complications resulting from vascular endothelial damage and from immune system deregulation, which contributes to acute respiratory distress and potentially to long term irreversible tissue damage. While the anti-inflammatory effects of low dose irradiation have been exploited for a long time in the clinics, few recent physiopathological and experimental data suggested the possibility to modulate the inflammatory storm related to COVID-19 pulmonary infection by exposing patients to ionizing radiation at very low doses. Despite level of evidence is only preliminary, these preclinical findings open therapeutic perspectives and are discussed in this article. On JanuCoronaviruses represent a large family of viruses that can cause a wide range of illnesses in humans, ranging from common cold symptoms to life-threatening SARS . SARS-CoMortality in COVID-19 patients is linked to a virus-induced “cytokine storm” . This isSevere forms of COVID-19 are preferentially observed in the elderly population, in people with underlying health problems such as diabetes and in those with deficit in their immune system . In seveThe C5a complement factor and its receptor (C5aR1) have key roles in the initiation and maintenance of inflammatory processes by recruiting neutrophils and monocytes, contributing to the pathophysiology of COVID-19 related acute respiratory distress syndrome. The levels of soluble C5a are increased in proportion to the severity of COVID19 infection. In animal models, inhibition of anti-C5aR1 axis prevented the C5a-mediated recruitment and activation of human myeloid cells in damaged lungs. These data open pharmacological perspectives for the modulation of COVID-19 related inflammation.Several recent physiopathological and experimental data suggest the possibility to modulate the inflammatory storm related to COVID-19 pulmonary fsfsfs by exposing patients to ionizing radiation at very low doses. Despite level of evidence is only emerging, these preclinical findings open therapeutic perspectives and are discussed in this article.The diagnosis of ARDS is conventionally based on well-defined parameters using the Berlin criteria, the oxygenation index and the Murray/lung Injury Score used by intensive care physicians to define the clinical, ventilatory, gasometric parameters  and radiological criteria to establish the diagnosis of this serious pulmonary syndrome and to adapt the ventilatory management as well as possible . RespiraChest scans are indicated to guide the management and monitoring of pulmonary symptoms in a patient with COVID-19. In addition to its use for early diagnosis, the chest scan has a prognostic role, making it possible to visually assess the extent of pulmonary lesions and monitor over time. The abnormalities observed on the CT scan are correlated to severity of clinical symptoms . AlthougDespite an unprecedented investment to look at therapeutic strategies, there is currently no effective treatment for COVID-19 infection. Most potential treatments have been evaluated in populations with significant heterogeneity and various levels of symptoms severity. Several existing antiviral treatments are being tested: remdesivir, combination lopinavir/ritonavir, combination lopinavir/ritonavir/interferon beta or even hydroxychloroquine. Remdesivir did not show effect in patients presenting with severe form of the disease, as assessed per mortality probability at day 28 . It neveThe long-term respiratory sequelae of COVD-19 are also a significant clinical concern. Based on data from 2003 SARS-CoV, showing that 35–60% of survivors developed pulmonary fibrosis with reduced lung function, it can be expected that at the end of this pandemic, a high number of patients surviving severe cases of Covid-19 will be severely affected by persistent respiratory complications. The true incidence of such late fibrosis in the COVID-19 context is however still uncertain . After AAs pointed out by Edward J Calabrese and Gaurav Dhawan, during the first half of the 20th century, radiation therapy was used a long time ago to treat pneumonia. Fifteen studies grouping together around 700 cases of pneumonia of bacterial origin (lobar and bronchopneumonia), including those unresponsive to treatment with sulfonamides, and described as being interstitial and atypical were treated effectively with low doses of X-rays, showing a decrease in clinical symptoms, and a lowering of mortality rates . Low dosHowever, the empiric beneficial effect of low doses of irradiation has been debated for over 50 years, in part because of the poor knowledge on the underlying mechanistic in the context of major concerns in terms of potential radiation-induced cancers . Indeed,Ex vivo) and in a preclinical study, using a viral pneumonia model (influenza A PR8 virus (H1N1). Authors showed that low doses of irradiation decreased both lung damages and inflammation and had no effect on viral expansion , increased tumor necrosis factor -beta (TNF-β), activation of several transcription factors, such as nuclear factor kappa beta (NFkB) and protein-1 (AP-1), apoptosis promotion, transforming growth factor - beta 1 (TGFβ1) activation, and stimulation of the activity of regulatory T cells . As revixpansion . These axpansion .In spite of these limitations, several prospective trials are currently being carried out in the context of the COVID-19 pandemic, encouraged by the lack of effective alternative and the high mortality probability in most severe cases of COVID-19 pneumonia . In addiAlthough numerous data show that low dose radiotherapy may have anti-inflammatory properties, the evidence supporting the use of low dose radiotherapy to treat COVID-19 infection remains preliminary. This approach could potentially have a favorable cost/effectiveness ratio, for a subgroup of COVID-19 patients for whom there is most often no therapeutic alternative and in a context of lack of access to resuscitation platforms . A prere


O-[2-(18F)-fluoroethyl]-L-tyrosine (FET) are increasingly used and provide valuable additional clinical information. We here report neuroimaging findings in a clincally symptomatic 53-year-old woman with a recurrent anaplastic oligodendroglioma with MRI findings highly suspicious for tumor progression. In contrast, FET PET imaging suggested treatment-related changes considerably earlier than the regression of contrast enhancement on MRI. In patients with oligodendroglioma, the phenomenon of symptomatic treatment-related changes is not well described, making these imaging findings unique and important for clinical decision-making.Following local and systemic treatment of gliomas, the differentiation between glioma relapse and treatment-related changes such as pseudoprogression or radiation necrosis using conventional MRI is limited. To overcome this limitation, various amino acid PET tracers such as  Treatment-related changes on conventional MRI are predominantly characterized by both an increase of contrast enhancement in the T1-weighted image and a hyperintensity in the fluid-attenuated inversion recovery (FLAIR) sequence, which either remain stable or even disappear over time without any change of anticancer treatment. Not infrequently, patients experience no clinical deterioration. Misinterpretation of these imaging findings may result in an unnecessary discontinuation of an effective treatment with a potentially negative impact on the patient’s survival. Due to the low specificity of conventional MRI, the differentiation of treatment-related changes from actual tumor progression in glioma patients following radiotherapy or chemoradiation using conventional MRI is challenging .18F)-fluoroethyl]-L-tyrosine (FET) in the care of glioma patients is gaining more attention. In recent years, a high diagnostic accuracy for the differentiation between tumor progression and treatment-related changes using FET PET has repeatedly been shown predominantly in patients with astrocytic gliomas  gene mutation and a 1p/19q codeletion and are histologically corresponding to the grade II or III of the in 2016 revised World Health Organisation (WHO) classification of Tumors of the Central Nervous System . This raWe here report the phenomenon of symptomatic treatment-related changes in a patient with an anaplastic oligodendroglioma.A 53-year-old female patient with a recurrent anaplastic oligodendroglioma  was diagnosed with the second local tumor recurrence 15 years after initial diagnosis. The first-line treatment included tumor resection and radiotherapy. Eleven years later, a first local tumor recurrence was treated with re-resection followed by adjuvant temozolomide chemotherapy. Four years later, the tumor recurred locally again. Following complete resection and re-irradiation, she underwent adjuvant nitrosourea-based chemotherapy with procarbazine and lomustine. After two cycles, the patient experienced a clinical deterioration with an expressive aphasia and a right-sided hemiparesis. According to current response assessment criteria Figure 1Following radiotherapy with or without alyklating chemotherapy, treatment-related changes such as pseudoprogression or radiation necrosis represent a major diagnostic challenge in patients with astrocytic glioma. In this group of brain tumors, the steadily increasing number of studies highlights the value of amino acid PET for the differentiation of treatment-related changes from actual tumor progression with a high diagnostic accuracy , 2, 4, 9On the other hand, oligodendrogliomas represent a small glioma subgroup and constitute only approximately 5% of primary intracranial tumors. Importantly, it has been demonstrated that in oligodendrogliomas the rate of treatment-related changes is significantly lower than in patients with gliomas without 1p/19q codeletion  , 11. ForAnother important aspect of the present case is the extensive increase of the FLAIR signal hyperintensity, suggesting non-enhancing tumor progression. In contrast, FET PET showed in spatial correspondence no pathological increased tracer uptake. Thus, FET PET seems also to be of value to rule out this particular phenomenon. In summary, this case highlights the value of FET PET for the diagnosis of treatment-related changes in a glioma subtype characterized by a naturally increased amino acid uptake and a low rate of treatment-related changes.The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.All procedures performed in the studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.Data acquisition, EB, NG, and J-MW. Writing of manuscript drafts, EB, NG, and J-MW. All authors contributed to the article and approved the submitted version.Supported by the Deutsche Forschungsgemeinschaft (DFG), project number 428090865 (EB and NG), and the Cologne Clinician Scientist Program (CCSP)/Faculty of Medicine/University of Cologne, funded by the DFG, FI 773/15-1 (J-MW).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


In the current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, testing for SARS-CoV-2-specific antibodies is paramount for monitoring immune responses in postauthorization vaccination and seroepidemiological studies. However, large-scale and iterative serological testing by venipuncture in older persons can be challenging. Capillary blood sampling using a finger prick and collection on protein saver cards, i.e., dried blood spots (DBSs), has already proven to be a promising alternative. However, elderly persons have reduced cutaneous microvasculature, which may affect DBS-based antibody testing. Therefore, we aimed to evaluate the performance of DBS tests for the detection of SARS-CoV-2 antibodies among nursing homes residents. We collected paired venous blood and DBS samples on two types of protein saver cards (Whatman and EUROIMMUN) from nursing home residents, as well as from staff members as a reference population. Venous blood samples were analyzed for the presence of SARS-CoV-2 IgG antibodies using the Abbott chemiluminescent microparticle immunoassay (CMIA). DBS samples were analyzed by the EUROIMMUN enzyme-linked immunosorbent assay (ELISA) for SARS-CoV-2 IgG antibodies. We performed a statistical assessment to optimize the ELISA cutoff value for the DBS testing using Youden's J index. A total of 273 paired DBS-serum samples were analyzed, of which 129 were positive, as assessed by the reference test. The sensitivities and specificities of DBS testing ranged from 95.0% to 97.1% and from 97.1% to 98.8%, respectively, depending on the population (residents or staff members) and the DBS card type. Therefore, we found that DBS sampling is a valid alternative to venipuncture for the detection of SARS-CoV-2 antibodies among elderly subjects.IMPORTANCE Since the implementation of newly developed SARS-CoV-2 vaccines in the general population, serological tests are of increasing importance. Because DBS samples can be obtained with a finger prick and can be shipped and stored at room temperature, they are optimal for use in large-scale SARS-CoV-2 serosurveillance or postauthorization vaccination studies, even in an elderly study population. In the current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, large-scale serological antibody studies are paramount to assess the true SARS-CoV-2 infection rate. Indeed, statistics on PCR-confirmed SARS-CoV-2 cases are far from ideal to estimate the true proportion of the population that has experienced a SARS-CoV-2 infection , as mildCapillary blood collection on protein saver cards after finger pricking using a lancet, the so-called dried blood spot (DBS), is a most valuable alternative because it is minimally invasive, can be performed at low cost, and has the potential for self-sampling and samples are easy to ship and store. DBSs are increasingly being applied as a minimally invasive alternative for infectious serological testing, especially in community- and population-based epidemiological studies, including for SARS-CoV-2 . Here, wIn four NHs, a total of 440 paired venous blood-Whatman DBS samples were obtained, of which 199 were from residents and 241 from staff members . The serThe numbers of true and false positive and negative results for the Whatman DBS and EUROIMMUN DBS samples, in comparison with the reference test, for all participants and categorized according to residents versus staff, are shown in P < 0.0001). The Pearson correlation coefficient for the reference test and the EUROIMMUN DBSs was 0.78  and significant (P < 0.0001) (the scatterplot was similar to that for the reference test versus the Whatman DBSs [data not shown]). A scatterplot showing the correlation between the OD values from the 150 paired Whatman and EUROIMMUN DBSs is depicted in P < 0.0001). For one sample pair, the Whatman DBS showed a positive result (OD of 1.41) but the EUROIMMUN DBS did not (OD of 1.02). However, the latter result is classified as borderline according to the manufacturer’s guidelines; this category was not considered in the current analysis, and this result was classified as negative.The scatterplot of the index values for the reference test (Abbott CMIA) and the OD values of the Whatman DBS ELISA is shown in P < 0.0001). The optimal cutoff point for the Whatman DBSs was found to be 1.14, which is almost exactly the value of 1.1 proposed by the manufacturer of the ELISA, and did not improve sensitivity or specificity. The optimal cutoff point for the EUROIMMUN DBSs was found to be 1.02, and applying this cutoff value improved both sensitivity (from 95.1% to 96.4%) and specificity (from 97.1% to 98.5%).To verify whether the ELISA Whatman DBS and EUROIMMUN DBS cutoff values could be optimized, we performed a receiver operating characteristic (ROC) analysis. The ROC curves are shown in In the present study, we evaluated the use of DBSs for detection of SARS-CoV-2 antibodies in residents and staff members from NHs. To the best of our knowledge, we report the largest clinical DBS validation study, comparing two different DBS samples (Whatman and EUROIMMUN) in a key target population in the current SARS-CoV-2 pandemic, namely, elderly persons residing in NHs.Overall, we demonstrated high sensitivity  and specificity  for DBS samples, compared to serum samples. Adjustment of the manufacturer-recommended cutoff value from 1.1 to 1.02 resulted in slight improvements in sensitivity and specificity for the EUROIMMUN DBS test only. We found no significant differences in sensitivity or specificity of the DBS tests between residents and staff members, for both Whatman and EUROIMMUN DBS, as demonstrated by the largely overlapping CIs. Furthermore, Whatman and EUROIMMUN protein saver cards yielded results that were in nearly perfect agreement.Several other studies in different populations, such as health care workers, key workers, athletes, and children, have evaluated DBS sampling for SAR-CoV-2 antibody testing –12. In t–10–Our findings show excellent diagnostic performance of DBS testing for both elderly patients and staff members from NHs. This supports the use of DBSs in large-scale SARS-CoV-2 serosurveillance studies as a valuable alternative to venipuncture, especially for elderly subjects, for whom venous blood can be challenging to obtain. In comparison to rapid serological tests, which can also be implemented in large-scale SARS-CoV-2 seroepidemiological studies, DBS tests offer higher sensitivity and specificity and the possibility of (semi-)quantitatively assessing the antibody response . MoreoveEspecially since the implementation of newly developed SARS-CoV-2 vaccines, serological studies are of increasing importance to fill existing research gaps. To date, the stability and duration of the antibody responses upon vaccination are unknown; however, it is important to determine whether annual boosting is needed. Furthermore, a correct antibody response cutoff value that refers to clinical immunity against SARS-CoV-2 is lacking. Additionally, it is of crucial importance to assess the effectiveness of the vaccines in the general population, which can differ from values measured in standardized clinical trials. In this context, the noninvasiveness of DBS testing, the minimal logistic constraints, the excellent test characteristics, and the possibility of quantitative assessment bring added value for conducting serosurveillance studies.Our study was limited by the use of the CMIA with venous blood samples, which assessed nucleocapsid antibodies and not spike antibodies, which were detected by means of ELISA with DBS samples. Conflicting results are available concerning the degree of persistence between antibodies directed against the nucleocapsid and spike antigens; however, it has been suggested that levels of IgG antibodies directed against the nucleocapsid antigen wane more rapidly postinfection than those directed against the spike antigen . NeverthThe current study was approved by the Ethical Committee of the Ghent University Hospital (reference number BC-07665) and was conducted according to the principles outlined in the Declaration of Helsinki. Each participant signed an informed consent form after being informed about the goal of the study and the sampling procedures. A confidential counselor, such as a nurse, signed for participants who were incapable of signing the consent form, such as residents with dementia, when consent was given by their legal representatives.In August 2020, before the start of the vaccination campaign, we contacted the management of four NHs within our network  and explained the goal of the study. We recruited NHs that had experienced a SARS-CoV-2 outbreak in the period of March to July 2020, in order to increase the likelihood of obtaining seropositive samples, thus minimizing the number of screened participants needed to obtain our calculated sample size. The management informed the families of residents and recruited a total of 199 residents and 241 staff members. All interested staff members and residents, except for residents with severe dementia (exclusion criterion), were eligible to participate in the study.We obtained approximately 5 ml of venous blood from each participant, in serum tubes, by venipuncture using a 23-gauge scalp vein set. Capillary blood was collected onto Whatman protein saver cards  after briefly puncturing the top edge of the distal phalanx of the middle or ring finger using 18-gauge safety lancets . EUROIMMUN protein saver cards  were marketed during sample collection and were evaluated together with the Whatman protein saver cards for a subset of participants. For each protein saver card, a preprinted circle was filled until saturated . In order to avoid sampling bias, for one-half of the participants , first a circle was filled on the Whatman protein saver card and then a circle was filled on the EUROIMMUN protein saver card; for the other one-half of the participants (those with an odd number), the opposite was done. Typically, an average of four blood drops were needed to saturate one circle. All blood collections were done under aseptic conditions. DBS samples were obtained by allowing the capillary blood to air dry on the protein saver cards for 1 h at room temperature.g for 8 min, and the tubes were stored at 4°C. The following day, serum was aliquoted into new serum vials and analyzed by means of the SARS-CoV-2 IgG Architect immunoassay (Abbott Laboratories). DBS samples selected for analysis (see below) were analyzed a maximum of 2 days after collection.Serum tubes were transported to the Laboratory of Clinical Microbiology of the Ghent University Hospital  within 6 h after sample collection. Upon arrival, serum tubes were centrifuged at 3,000 × All serum samples were analyzed for antinucleocapsid SARS-CoV-2 IgG serological results by using the Architect i2000sr Plus system (Abbott). This system allows high-throughput screening of the sera, providing real-time information on the number of positive samples. This way, we could rapidly validate DBS tests for our ongoing and future studies in NHs. The Architect i2000sr Plus system uses the CMIA technique to detect antibodies. After thawing the sera and vortex-mixing them briefly, the Architect system analyzes the samples automatically using a SARS-CoV-2 assay, a specific calibrator kit, and a specific control kit. We used an in-house-validated cutoff index of 0.9 to classify sera as positive (≥0.9) or negative (<0.9) for SARS-CoV-2 IgG antibodies.The DBS samples were analyzed for the presence of antispike (S1 antigen) SARS-CoV-2 IgG antibodies by means of ELISA  and not the Abbott CMIA because the latter assay requires a volume of 150 μl, which is more than the volume of capillary blood absorbed on one circle of the DBS card. One circle (6-mm diameter) was cut out from each DBS card using a puncher and was placed in a well of a sterile 96-well U-shaped microtiter plate. To avoid cross-contamination, the puncher was cleaned between punches using a 70% alcohol solution and a cotton swab. A total volume of 250 μl preheated (1 h at 37°C) sample buffer was added to each sample well of the 96-well microtiter plate, and the plate was incubated at 37°C for 1 h. After gentle mixing of the eluate by means of up-and-down pipetting, a total of 100 μl of this eluate was used for ELISA, according to the manufacturer’s instructions. The ELISA was run on the automated Behring Elisa Processor III . DBS samples were classified according to their antibody index OD value (450 nm) as negative (<1.1) or positive (≥1.1), as recommended by the manufacturer. The borderline category was not considered.The sample size was calculated using the methodology described by Buderer, focusing on the sensitivity . Here, wThe sensitivity and specificity of the DBS tests were calculated using the Abbott CMIA with sera from venous blood samples as a reference test. The 95% CIs were calculated using the Wilson-Brown method .To determine the optimal cutoff value for the Whatman and EUROIMMUN DBS samples, we calculated Youden’s J index. The accuracy was calculated using the AUC of the ROC curves. All analyses were performed using the statistical software GraphPad Prism 6 .


C. elegans declines with age. This decline is the cumulative result of age-related changes in several steps of germline function, many of which are highly accessible for experimental investigation in this short-lived model organism. Here we review recent work showing that a very early and major contributing step to reproductive decline is the depletion of the germline stem and progenitor cell pool. Since many cellular and molecular aspects of stem cell biology and aging are conserved across animals, understanding mechanisms of age-related decline of germline stem and progenitor cells in C. elegans has broad implications for aging stem cells, germline stem cells, and reproductive aging.Like many animals and humans, reproduction in the nematode  C. elegans is a well-established model for germline development and for aging. Several interesting related areas of study include how the C. elegans germ line regulates longevity  into which contents flow proximally to supply growing oocytes. As many as 85% of adult germ cells that enter prophase of meiosis I have been estimated to later undergo apoptosis in late pachytene. This cell death is thought to constitute a nurse cell function providing material for growing oocytes via the core.Adult germ cells that do not undergo programmed cell death continue to mature as oocytes, arresting in diakinesis while awaiting their “turn” to reach the proximal-most position in the oviduct . In respC. elegans germline stem cell identity depends on canonical Notch signaling. Each distal end of the hermaphrodite gonad is capped by single somatic cell called the distal tip cell  declines with age. This occurs in both unmated and mated hermaphrodites despite the profound gene expression changes that accompany sperm depletion . In bothdaf-2, which encodes the sole insulin/IGF-like receptor in C. elegans, promotes aging, a role was postulated for this gene and its signaling pathway (hereafter abbreviated as IIS) in reproductive aging. Reduction-of-function (rf) mutations in daf-2 extend lifespan in a manner dependent upon the transcription factor DAF-16 FOXO  in the intestine of the worm partially reverses the effects of daf-16 loss on lifespan of the daf-16; daf-2 double mutant, making it more similar to daf-2 alone  in muscle or neurons influence age-related PZ loss in the daf-16; daf-2 double mutant  is required in certain cells of the proximal somatic gonad (PSG), a source of DAF-16 not previously linked to longevity nor to PZ regulation. Specifically, expression of daf-16(+) in a subset of (fos-1a-positive) PSG cells of the spermatheca and uterine lineage completely reversed the effect of the daf-16 mutant such that the double mutant daf-16; daf-2 expressing daf-16(+) in PSG cells maintained a larger PZ pool with age, similar to the daf-2 mutant alone. Consistent with this finding, tissue-specific RNAi depletion of daf-16 in the same PSG cells mimicked loss of daf-16, while depletion of daf-16 in the germ line did not  activity in promoting longevity. However, this possibility was ruled out since lifespan is unaffected in daf-16; daf-2 double mutant worms bearing the PSG-expressed daf-16(+) transgene. Thus, age-related germline progenitor pool decline and organismal lifespan can be modulated by the same IIS signaling pathway, but from anatomically distinct activities of daf-16 in the worm  is itself subject to nutritional state   . A bona Cell cycle analysis in mated hermaphrodites, measuring M and S phase index as well as G2 duration and total non-S duration, indicated that between days 1 and 3 of adulthood, the duration of the G2 is extended ∼2 fold (2.5–4.9 h), as is the entire non-S portion of the cell cycle . Interestingly, neither the M-phase index nor S-phase index change over this timeframe in mated worms. One possible explanation is that pro-metaphase may be lengthened as well, as these studies used anti-phospho-histone H3 to measure mitotic index. Taking all these data into account, the average speed of the cell cycle is reduced ∼2-fold by day 3 .In addition to PZ cells lengthening certain cell cycle stages in aging mated worms, the rate at which they enter meiosis is also reduced, as indicated by pulse-chase analysis of EdU-labeled germ cells . In geneIn addition to a slower rate of entry into prophase of meiosis I, progress through meiotic prophase also slows with age. Aging extends time spent in leptotene/zygotene as well as pachytene . HoweverAmong a number of germline aging phenotypes, the age-related reduction of the PZ pool occurs in essentially all individuals within the aging population, both mated and unmated hermaphrodites . In addiC. elegans  cells in the distal germ line. One possibility is that this conserved Notch signaling pathway is affected by age-related cellular and tissue-level changes. Since Notch activity regulates stem cell fate in several different mammalian organ contexts, worm studies may inform our understanding of stem cell aging in higher organisms as well. Even in cases where other (non-Notch) signaling pathways control stem cell fate, features of stem cell signaling in the context of aging tissues may well be conserved.C. elegans germ line occurs via membrane-bound ligand expression in the somatic niche cell and the GLP-1 receptor on the surface of germ cells. Why does the number of SYGL-1-expressing stem cells decrease as animals age? Are the levels of Notch signaling in the stem cells reduced? If so, at what step of the signaling pathway? Is the post-transcriptional regulation of Notch pathway components or effectors different in old worms than in young ones? How do changes in Notch signaling interface with the changes in the germ cell cycle? To what extent is the reduction in Notch signaling due to general age-related changes to cell membranes or signaling centers? Is the efficacy of signal reception or transmission affected by age-related alterations in receptor membrane localization or receptor processing? Is the reduction in Notch signaling due to aging in the soma or in the germ line? Do age-related changes in nuclear transport or germ cell chromatin interface with transcriptional activation downstream of receptor signaling? Do changes to Notch pathway signaling reflect interactions with other age-related signals that impinge on the germ line? If so, how?Notch activation in the Equally interesting will be an understanding of how specific aspects of cell biological aging, such as changes to the basement membrane, to mitochondrial function, or to proteostasis, contribute to the decline in stem cell fate and function. It is possible, for example, that some aspects of cellular aging impact cell cycle while others impact cell fate decisions via effects on specific signal transduction pathways.daf-16 expression in a subset of proximal somatic gonadal cells regulate the rate of decline of the PZ pool? One hypothesis is that a daf-16-dependent secreted factor signals directly to the germ line. Alternatively, PSG-to-germline communication could occur through intermediate tissues. The relevant targets of daf-16 in the PSG will also be important to identify. To what extent does this pathway act at the level of germline stem cell identity, cell cycle, or both? It will also be of interest to determine how other age-defying conditions and genetic manipulations influence PZ aging, and, if so, whether they act by mechanisms similar to or distinct from the daf-2 pathway.With respect to IIS, organismal lifespan and the germline progenitor pool are regulated in anatomically distinct ways. How does daf-16 in the proximal somatic gonad , pheromone, and temperature—impacts many aspects of development. A well-understood example is the regulation of dauer, an alternate pre-reproductive larval stage that is resistant to poor environmental conditions (see ions see , and IISions see .C. elegans. Under extreme conditions of starvation in late larval stages, reproductive adults can arrest germline stem cell divisions and slow the rate of oocyte production from the usual ∼3 oocytes ovulated per hour per gonad arm in peak times to fewer than one every 8 h (Aging and reproduction are particularly sensitive to environmental manipulation in very 8 h . Remarkavery 8 h . ResponsSlowing the germline progenitor cell cycle during environmentally unfavorable conditions or after sperm depletion may be an evolutionary strategy to allow sperm-depleted hermaphrodites to temporarily retain germline progenitors despite advancing age, such that PZ cell proliferation, entry into and through meiotic prophase, and oocyte production can resume quickly upon locating new food sources and/or males with which to mate.C. elegans life history and reproductive strategies have been sculpted by evolution to ensure its success in the wild. Nevertheless, together with its advantages as a laboratory model organism, these quirks offer rich opportunities for experimental analysis of reproductive and stem cell aging. Given the conservation of cellular and molecular processes from worms to mammals, and given the history of what model organism research has contributed to fundamental biology relevant to human health, studies on germline stem cell aging should offer broad implications beyond the worm.The quirks of Both authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Developing a vaccine against COVID-19 is considered a key strategy to end the pandemic. However, public acceptance is reliant on beliefs and perception toward the vaccine. Therefore, the study aimed to assess the beliefs and barriers associated with COVID-19 vaccination among the Saudi population.An online self-administered questionnaire was distributed across the main regions of Saudi Arabia on May 2020. The questionnaire addressed the socio-demographic variables, beliefs toward COVID-19 vaccination, and potential barriers that may prevent participants from being vaccinated. The association between COVID-19 vaccine acceptance and sociodemographic variables were analyzed. Logistic regression analysis was used to identify the predicting variables of vaccine acceptance.Out of 3101 participants, 44.7% were accepting of COVID-19 vaccination if available, whereas 55.3% admitted hesitancy. Younger, male, who received seasonal influenza vaccine were more likely to accept taking the vaccine. The study found that concerns about side effects were the key barrier for vaccine acceptance. Furthermore, the majority of refusers may accept the vaccine if additional studies confirmed safety and effectiveness.Results can be utilized in planning vaccination campaigns while waiting for vaccine development.The online version contains supplementary material available at 10.1186/s12889-021-11501-5. The coronavirus disease-19 (COVID-19) is highly contagious and caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) . VaccinaA recently published review  demonstrThe study is cross-sectional in nature and was conducted on May 2020. A validated, self-administered electronic questionnaire was distributed online through social network sites, such as WhatsApp and Twitter.The study targeted potential participants from the main five regions in Saudi Arabia to attain results that would be generalizable across the country. The questionnaire has been designed and developed for the purpose of this study  dated May 19, 2020.Data cleaning and analysis were performed using the Statistical Package for Social Sciences (SPSS) v. 24.0 . Descriptive statistics including frequencies (n) and percentage (%) were used to present the demographic data of the participants.For the second domain, responses were rated from 0  to 3 . The points from each question were summarized to calculate the total score. A total score of > 2 was considered positive belief, whereas the opposite is true for a total score of < 2.P-value < .05 was considered statistically significant.Chi-square test was used to assess the significance of the association (contingency) between COVID-19 vaccine acceptance and sociodemographic variables. Logistic regression analysis was used to calculate the odds ratio (OR) and 95% confidence intervals (95% CI) to identify the predicting variables of vaccine acceptance. A A total of 3101 participants from five regions in Saudi Arabia were recruited. Table Table The same pattern of beliefs was found among a high-risk group who was defined on the following criteria: > 60 years old, healthcare worker, working with foreigners, working in catering, or having chronic diseases . A remarkable increase in the likelihood of being vaccinated was observed for those who held positive beliefs. In this case, the participants with positive beliefs toward COVID-19 vaccination were 9.288 times more likely to accept vaccination if available (95% CI: 7.72–11.17).P-value > 0.05).However, Table Table However, more than two-third of vaccine refusers (63.9%) indicated that they will agree to be vaccinated if further studies confirmed the safety and effectiveness of COVID-19 vaccination. A total of 44% of vaccine refusers will agree to vaccination if made compulsory by the government but only 11% if made compulsory by their jobs not the government. Furthermore, one-fifth of vaccine refusers may accept vaccination if recommended by physicians. A significant percentage of vaccine refusers (17.8%) will not take the vaccine under any of the cited circumstances.Results indicated that the sample population was divided between vaccine acceptance and refusal whenever available. This finding highlights the dilemma of the topic in the Saudi population, where half of the population may accept vaccination. Moreover, the study revealed several key predictors of hesitancy toward COVID-19 vaccination. Older females with high levels of education, no history of influenza vaccine uptake, and negative beliefs toward vaccination were more likely to display hesitation toward COVID-19 vaccination. Notably, being part of a high risk group did not improve the odds of being vaccinated. The most significant predictor of vaccination is holding positive beliefs.Although studies that assessed hesitancy toward COVID-19 vaccination are limited, the acceptance/ hesitancy rates toward any vaccine are diverse across the world . CorrespThe most significant predictor for acceptance of COVID-19 vaccination was beliefs. This result is congruent with that of a previous systematic review conducted by Bish et al.  to assesResults demonstrated that self-reported influenza vaccine uptake was a positive predictor for the acceptance of eventual COVID-19 vaccination. Similarly, previous studies have shown that the rate of H1N1 vaccine acceptance was higher among participants with a history of uptake of the seasonal influenza vaccine in the USA  and FranMale gender was another positive predictor for acceptance of COVID-19 vaccination. This result could be due to the reported high rates of COVID-19-related morbidity and motility among male infected patients . FurtherAnother positive predictor for the acceptance of COVID-19 vaccination was age. Younger participants tended to be more accepting of vaccination in contrast with older participants. The same trend was observed among participants with secondary and university levels of education compared to participants with higher education. A possible explanation is that younger participants are more frustrated with social restrictions and curfews associated with the COVID-19 crisis and would thus be more willing to be vaccinated. At the same time, younger people may be more accustomed and trusting of science and technology in contrast with their older counterparts. At the same time, school suspension may negatively affect the academic performance of school-aged and university participants. Therefore, they are more impatient to bring an end to the situation and thus more accepting of vaccination. Further studies should explore these possibilities as these data can be useful for future vaccination campaigns.Although a previous study indicated that the majority of Chinese healthcare workers were willing to accept COVID-19 vaccination , more thAs stated by MacDonald , factorsAnother barrier to COVID-19 vaccination was the COVID-19 conspiracy theory, which has spread very rapidly around the world , 28 via The widespread conspiracy theory could be due to the people’s psychological need to understand the unexpected events associated with the COVID-19 pandemic . MoreoveIn agreement with previous research in China , the majNotably, during the H1N1 influenza A pandemic, the public acceptance rate of Americans toward vaccination before its approval was 8.7% . HoweverGiven that vaccination is the cornerstone of reduced healthcare burden caused by the COVID-19 pandemic, the results of the study can be utilized for planning evidence-based vaccination campaigns while waiting for vaccine development . By enhaThe current study has certain limitations. The study was conducted using an online self-administered questionnaire instead of face-to-face interviews due to the implemented curfew and social distancing restrictions during the COVID-19 pandemic. As a result, reporting bias should be considered. Moreover, the cross-sectional study represents public acceptance and beliefs toward COVID-19 vaccination during the pandemic before the availability of a vaccine. With this notion, people’s acceptance and beliefs could be changed with time as reported in other studies on pandemics .Furthermore, disparities were noted across regions with regard to response rate. The highest response rate was noted for the Western region, which is one of the largest in the country. Makkah, AlMadinah, and Jeddah cities can be found in this region, which are the most affected cities by COVID-19 in Saudi Arabia. This scenario could make people from the region more aware and anxious about topics related to COVID-19. Other factors may have contributed, such as availability of the Internet as well as differences in perception toward the use of social media platforms across regions.Results from current study can be utilized in planning vaccination campaigns to increase the public awareness.Additional file 1: Supplement 1. English version of the questionnaire. Supplement 2. Barriers associated with acceptance of COVID-19 vaccine among high risk group (N = 882).


Saposin C (sapC) is a lysosomal, peripheral-membrane protein displaying liposome fusogenic capabilities. Proteoliposomes of sapC and phosphatidylserine have been shown to be toxic for cancer cells and are currently on clinical trial to treat glioblastoma. As proof-of-concept, we show two strategies to enhance the applications of sapC proteoliposomes: (1) Engineering chimeras composed of sapC to modulate proteoliposome function; (2) Engineering sapC to modify its lipid binding capabilities. In the chimera design, sapC is linked to a cell death-inducing peptide: the BH3 domain of the Bcl-2 protein PUMA. We show by solution NMR and dynamic light scattering that the chimera is functional at the molecular level by fusing liposomes and by interacting with prosurvival Bcl-xL, which is PUMA’s known mechanism to induce cell death. Furthermore, sapC-PUMA proteoliposomes enhance cytotoxicity in glioblastoma cells compared to sapC. Finally, the sapC domain of the chimera has been engineered to optimize liposome binding at pH close to physiological values as protein–lipid interactions are favored at acidic pH in the native protein. Altogether, our results indicate that the properties of sapC proteoliposomes can be modified by engineering the protein surface and by the addition of small peptides as fusion constructs. The protease-resistant, heat-stable, lysosomal protein saposin C (sapC), is a peripheral-membrane protein with important roles in lipid degradation  and lipiSapC shows special characteristics, such as high stability and protease resistance, liposome fusogenic activity and pH-dependent liposome binding. In addition, sapC can be over-expressed by recombinant methods and purified by standard chromatographic techniques. Altogether, these attributes point to sapC as an attractive candidate for biotechnological applications and biologics design. In fact, proteoliposomes formed by sapC and liposomes of phospholipid 1,2-dioleoyl-sn-glycero-3-phosphatidylserine (DOPS) have been shown to promote apoptosis in cancer cells ,12. It hWith the aim of enhancing the properties and applications of sapC proteoliposomes, we have designed as proof-of-concept a protein chimera carrying sapC at the N-terminus and the active domain (BH3 domain) of the Bcl-2 proapoptotic protein PUMA at the C-terminus. Hence, we refer to the chimera as sapC-PUMA. In addition, we have modified the electrostatic surface of sapC in the chimera to improve liposome binding for potential applications at physiological pH.BH3) suffices for promoting cell death. BH3-derived peptides and peptidomimetics are intensively investigated as anticancer drugs -SOFAST-HMQC experiments  is the total concentration of liposomes, KD is the dissociation constant for protein-liposome binding and n is the Hill coefficient indicating the number of adsorbed molecules per binding site .Liposome titrations were done using -sofast-HMQC experiments -2D NMR that the saposin fold was not perturbed by the presence of the mutations in the sapC domain. Dapp ~1.6 nM at pH 6. Thus, the affinity of sapC-PUMA-DM for liposomes has almost doubled relative to wildtype sapC-PUMA (The mutant chimera (sapC-PUMA-DM) in which the electrostatic surface of the sapC domain is more positively charged due to mutations of Asp 55 and Glu 67 to Arg, is expected to bind with increased affinity to liposomes. We first checked using 7], and d1H NMR spectrum when the pH is decreased from 6.8 to 4.2 (conditions necessary for abundant liposome fusion)  . In contWe have used dynamic light scattering to study liposome fusion orchestrated by sapC, sapC-PUMA, and sapC-PUMA-DM  and its time dependence. For sapC, we observe that liposomes increase in diameter from ~100 nm to ~400 nm in approximately 5 min after protein addition and reach approximately 900 nm after 200 min a. After BH3 peptide and non-conservative mutations in sapC. Potentially, this fusogenic effect could be leveraged to design cell penetrating capabilities of biologics based on sapC and sapC mutants.We investigated the effect of 150 mM NaCl on liposome fusion mediated by sapC obtaining similar results, which indicates that salt does not play an important role in liposome fusion b. FurtheAssemblies of sapC with DOPS liposomes have been shown to be highly cytotoxic for a variety of cancer cells and in particular very effective in killing glioblastoma ,12. SapCBH3 domain. For this purpose, we followed the preparation of proteoliposomes described in the original assays [Our biophysical studies demonstrate that the sapC chimeras retain the function of both sapC and PUMA at the molecular level. To further test this function, we performed viability assays in glioblastoma cells in the presence of proteoliposomes carrying sapC, sapC-PUMA, and sapC-PUMA-DM with the expectation to observe similar cytotoxicity for sapC proteoliposomes as previously reported  and incrl assays .BH3 peptide. A killing activity ratio defined as the percentage of cells killed by sapC-PUMA relative to sapC proteoliposomes, indicates that cytotoxicity is enhanced approximately 40% by sapC-PUMA for certain concentration values  in the chimera are still functional at the molecular level and that the engineered proteins show, as expected, increased cytotoxicity due to an additive effect of the PUMA peptide. SapC-PUMA retains the saposin fold and sapC functionality by exhibiting reversible pH-dependent binding to liposomes and liposome fusogenic capability. In addition, the PUMABH3 component of the chimera binds tightly to Bcl-xL, its natural antagonist, in the presence of the sapC domain. These results indicate that both sapC and PUMABH3 have minimal interference in each other’s function, thus fulfilling the basic requirement of the chimera design.We show by NMR that both domains (sapC and PUMAPrevious work reported that maximum binding of sapC to liposomes occurs upon acidification . Here weBy performing biochemical and biophysical studies, we have determined that both domains of the chimera protein retain proper fold and binding capabilities, which gives greater confidence to the effectiveness of the chimera constructs prior to performing in vitro cell studies. Importantly, our viability studies indicate increased cytotoxicity for sapC-PUMA compared to sapC proteoliposomes. This is a promising result showing that potential anticancer peptides can be added to sapC to elicit additional cytotoxicity. Because sapC-DOPS is currently on clinical trial to treat refractory glioblastoma , an incrAltogether, our results indicate that sapC is a good candidate for engineering chimeric biologics by adding peptides or small proteins as potential therapeutics. Future work is necessary to test the effect of other therapeutic peptides/proteins in sapC chimeras at the molecular and functional levels in the presence and absence of liposomes or lipid nanoparticles, and to further tune liposome binding affinity with additional mutations to target specific tissues and promote liposome fusion under different pH conditions.


Introduction: It has been suggested that post-transoral robotic surgery (post-TORS) radiotherapy (RT) might reduce the dose to organs at risk (OARs) adjacent to the primary tumor bed; however, the evidence supporting this has yet to be sufficient. This study examined the radiation dose reduction to OARs by omitting the primary tumor bed through the use of an Auto-Planning (AP)-based workflow.Methods: Twelve patients were identified who underwent post-TORS RT to the primary tumor bed and the unilateral/bilateral neck lymph nodes. In each patient, two treatment plans were designed: a Comprehensive (Comp)-plan treating the original planning target volume (PTV) including both the primary tumor bed and the lymph nodes, and a Neck-plan treating only the lymph nodes and omitting the primary tumor bed. Both plans were optimized using AP to ensure plan quality consistency. We compared the doses received by 95% of the primary tumor beds and lymph nodes (D95%) and our institutional dose constraints for the OARs between the Comp- and Neck-plans. Statistical analysis was performed using R Statistical Software  with a two-tailed paired Wilcoxon signed-rank test.Results: All plans met target dose coverage requirements with at least 95% of the PTVs covered with the corresponding prescription doses. The primary tumor bed in the Neck-plans was spared with a significantly lower mean D95% . The mean dose to the oral cavity  and the supraglottis  was significantly lower in the Neck-plans.Conclusion: This study suggests that sparing the primary tumor bed during post-TORS RT offers dosimetric benefits to nearby OARs with significant dose reductions to the oral cavity and supraglottis. Further study of the clinical risks and benefits afforded by this strategy is needed. In recent years, the incidence of oropharyngeal squamous cell carcinoma (OPSCC) has increased, possibly as a result of human papillomavirus infection (HPV), and is estimated to double in the United States by 2030 . TransorInspired by recent studies to quantify the dose reduction to the OARs when excluding the primary tumor bed from the target volume, we adopted a comprehensive (Comp) method using the Auto-Planning (AP) technique to evaluate the potential dosimetric benefits of omitting the primary tumor bed from the target volume of the postoperative RT after TORS ,7.From an IRB-approved registry, patients diagnosed with HPV-positive pathological stage T1/2 base-of-tongue (BOT) OPSCC with at least one positive node who underwent TORS followed by postoperative RT between 2009 and 2015 at our institution  were retrospectively identified. The primary tumor bed and the ipsilateral neck lymph nodes were delineated as the high-risk clinical target volume (CTV), and the contralateral nodes as the low-risk CTV. The high- and low-risk planning target volumes (PTVs) were generated by adding a 2.5-mm uniform margin to the corresponding CTVs, and were originally treated with 60 Gy and 54-56 Gy, respectively, using simultaneous integrated boost RT. Each patient was then subsequently re-planned for two different treatment plans: one (Comp-plan) duplicating the original RT plan by treating the same comprehensive PTVs, and the other (Neck-plan) only treating the high- and low-risk neck lymph nodes with the aforementioned dose prescriptions, while the primary tumor bed was excluded from the original high-risk PTV.The Comp- and Neck-plans were generated on a Varian TrueBeam linear accelerator  with Millennium multi-leaf collimators . The volumetric modulated arc therapy (VMAT) technique with two 360° arcs of 6 MV photon beams was used in both plans. The Auto-Planning module in Pinnacle 9.10  was used for plan optimization. The principles of AP have been detailed in previous publications ,9. In suAP has been extensively used for various sites in our department with consistently reasonable results. The AP optimization objectives used in this study were determined based on our prior experience in planning and treating for OPSCC ,11. To aFollowing the institution protocol for OPSCC plans, the coverage to the primary tumor bed, the mean dose to the oral cavity, pharyngeal constrictor, supraglottis, larynx, ipsi-/contra-lateral parotid and submandibular, and the max dose to the mandible and the spinal cord were evaluated and compared between the Comp- and Neck-plans. Statistical analysis was performed using R Statistical Software  with a two-tailed paired Wilcoxon signed-rank test. A p-value less than 0.01 was considered statistically significant.Twelve patients with BOT OPSCC were included in this IRB-approved study. Of these, seven (58%) had T1 and five (42%) had T2 tumors. Six lesions were primarily left-sided and six were primarily right-sided. Bilateral neck-lymph nodes were treated in nine patients (75%) and three patients (25%) were treated unilaterally. Routinely, the CTV included anatomic levels II-IV of the neck, and in select cases included levels IB and V depending on tumor factors such as nodal burden or location of the primary. The contralateral neck was routinely treated for cancers with significant BOT involvement.All Comp- and Neck-plans were successfully generated with the AP technique, and reviewed and approved by radiation oncologists. Dosimetric comparisons of the primary tumor beds and the OARs are summarized in Table Figures This study examined the dosimetric advantages of avoiding the primary tumor bed in postoperative RT for patients treated with TORS for their OPSCC. By excluding the primary tumor bed from the target volume, any OARs adjacent to the primary tumor bed received less radiation dose, which may reduce the likelihood of radiation-induced toxicities. Because of the large variations in plan qualities for head and neck radiotherapy planning, the extent of dose reductions in the OARs, as well as the primary tumor bed, have been inconsistently reported. In this study, we used a consistent planning methodology with the Auto-Planning technique to minimize the variations in plan quality. Despite not specifically considering the primary tumor bed as an avoidance structure, we spared the primary tumor bed significantly compared to the other studies -7.Fried et al. first presented the concept of sparing the primary sites in their pilot study . PossiblA more recent study by Lazarev et al. was conducted in a similar fashion but with a larger sample size of 21 patients . This stIn this study, we presented a more comprehensive methodology to evaluate the dosimetric benefits of sparing the primary tumor bed. We used the AP technique to minimize the variations in the plan qualities. In theory, the dose distribution of a radiotherapy plan created using AP should solely depend on the anatomy of the plan, such as the location of the PTV and the relative geometries of an OAR to the PTV. Because of the identical anatomy between the Comp- and Neck-plans except for whether the primary tumor bed was included in the PTV or not, it was expected that any differences in the dose distribution between the two plans should only be found in the vicinity of the primary tumor bed, whereas the dose distribution far away from the primary tumor bed should be comparable. Therefore, by eliminating any potential differences in plan qualities, we were able to conduct valid comparisons to demonstrate the dosimetric benefits of sparing the primary tumor bed.Notably, the dose to several OARs in our Comp- and Neck-plans were lower than those reported in the previous two studies, which also suggested better plan qualities achieved using the AP technique, as also observed in the previous literature . While iIn addition, dosimetric sparing of the primary tumor bed was verified in this study. The dose to the primary tumor bed significantly decreased in the Neck-plans . These dose reductions confirmed the purpose of excluding the primary tumor bed. It should be noted that, for the purpose of valid comparisons between the Comp- and Neck-plans, we did not intentionally minimize radiation dose to the primary tumor bed in Neck-plans, nor place any optimization objectives to the primary tumor bed. Instead, the same set of Auto-Planning optimization objectives was used in both the Comp- and Neck-plans to avoid any biased comparison.Based on the significant dose reductions to the supraglottis and the oral cavity in the present study, it is expected that excluding the primary tumor bed during adjuvant RT after TORS would provide meaningful clinical benefits. It has been long observed and verified by multiple studies that the occurrence of radiation-induced dysphagia is strongly correlated with the mean dose to the supraglottis ,14. MoreHowever, omitting the primary tumor bed from postoperative RT could also have negative consequences as it might also increase the risk of recurrence. Using the AP technique in our study, the mean doses to the primary tumor bed were approximately 30 Gy. This dose might be insufficient to treat residual cancer cells in the tumor bed. However, recently published studies on the omission of the primary tumor bed for patients with resected OPSCC reported higher doses to the primary tumor bed (40-43 Gy in  and 37 GExcluding the primary tumor bed from the target volume of postoperative RT after margin negative TORS for OPSCC appears to be dosimetrically beneficial with a lower radiation dose to the oral cavity and supraglottis. These dose reductions could be potentially translated into a lower incidence of radiation-induced toxicities.


The novel coronavirus outbreak started in December 2019 and rapidly spread around the globe, leading to a global pandemic. Here we reported the association of microbial agents identified in oropharyngeal and nasopharyngeal samples from patients with SARS-CoV-2 infection, using a Pan-microarray based technology referred to as PathoChIP. To validate the efficiency of PathoChIP, reference viral genomes obtained from BEI resource and 25 SARS-CoV-2 positive clinical samples were tested. This technology successfully detected femtogram levels of SARS-CoV-2 viral RNA, which demonstrated greater sensitivity and specificity than conventional diagnostic techniques. Simultaneously, a broad range of other microorganisms, including other viruses, bacteria, fungi and parasites can be detected in those samples. We identified 7 viral, 12 bacterial and 6 fungal agents common across all clinical samples suggesting an associated microbial signature in individuals who are infected with SARS-CoV-2. This technology is robust and has a flexible detection methodology that can be employed to detect the presence of all human respiratory pathogens in different sample preparations with precision. It will be important for differentiating the causative agents of respiratory illnesses, including SARS-CoV-2. Viral pandemics that emerged over the last four decades were initiated across different geographic regions of the world and these includes the human immunodeficiency virus (HIV), severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) coronaviruses, influenza H1N1 virus, Ebola virus, Zika virus and most recently, the pandemic caused by SARS-CoV-2 which was initiated in Wuhan, China . These vThe SARS-CoV-2 (the associated disease is known as COVID-19) was first reported in China in December 2019 , and it DNA Endonuclease-Targeted CRISPR Trans Reporter (DETECTR)  . This teDETECTR) .We now report the development of a DNA microarray-based technology for detection of SARS-CoV-2 in patient samples. In previous studies we utilized this technology to report identification of a distinct microbiome in tumor samples from patients with different cancers, which we refer to as PathoChIP . We wereWe have developed this technology as a potential resource that can be used as a sensitive diagnostic tool for early detection of highly infectious microorganisms in clinical samples, which includes all known respiratory viruses as well as SARS-CoV-2. DNA and RNA were isolated from nasopharyngeal (NP) and oropharyngeal swabs (OP), which were then subjected to whole transcriptome amplification (WTA) where the amplified products were used to hybridize against probes on specifically designed PathoChIPs containing sequences that are unique, conserved and frequently mutated across various regions of the SARS-CoV-2 genome. The current version of the PathoChip (V5) contains 60,000 probes and represents all known viruses, including SARS-CoV-2, and pathogenic microbes that comprise 250 helminths, 130 protozoa, 360 fungi, and 320 bacteria, totaling more than 6,000 accessions .For SARS-CoV-2 detection, there are a total of 19 probes spanning the genome, consisting of 10 unique, 6 conserved, and 3 located in highly mutated regions, which are printed on the arrays. The arrey is also flexible in that new probes can be added and printed allowing for detection of new variants or new agents in a matter of days. Furthermore, the PathoChIP screening technology includes an amplification step that allows for detection of microorganisms and viruses that are present in low copy numbers or as fragmented genomes within samples. Different sample types can also be used, including swabs, tissues, cells, blood and other body fluids. Thus, PathoChIP allows a wide range of clinical samples, of various types, to be screened with high sensitivity and with relatively rapid detection of SARS-CoV-2 and other present microbial agents. Furthermore, as this technique relies on the hybridization of both microbial DNA and RNA, the sensitivity and the accuracy are far greater than the current conventional techniques employed. A more detailed description of the technology was previously described in multiple published studies .GSE166281) showed a strong hybridization signal intensity (HSI) across all six probes within the conserved regions, the three probes within the frequently mutated regions, and the ten probes within the unique regions of the genome and were nicely saturated across all probes present  from SARS-related Coronavirus-2, obtained from BEI resources  which wa present . Further present . More sp present . The resTo determine the specificity of the probes, we used a sample NR-52358 (obatined from BEI resources), which is a synthetic RNA mixture containing fragments from the SARS-CoV-2 genomic regions encoding parts of ORF1ab, N and E. Our screen revealed that PathoChip consistently detected strong HSI for selected probes 1, 2 and 6 that lies within these unique regions of SARS-CoV-2 genome as expected . As the 7 to 8 RNA molecules. The results showed a trend of consistently decreasing average HSI for all nineteen SARS-CoV-2 probes. The signals plateaued at an input of approximately 700 genomic RNA molecules (To determine the lower limit of detection (LLOD) for the SARS-CoV-2 probes on PathoChIP, we serially diluted the positive control genomic RNA reference sample, NR-52285, to a dilution where the viral RNA copy number was less than 10. This was diluted with decreasing 10-fold concentrations of input to picograms levels , which rolecules . Furtherolecules  measuredolecules , and inpolecules . This maolecules .A total of eight OP patient swab samples were screened, with six samples detecting an average HSI for unique SARS-CoV-2 probes consistently within a range of 0.50 and 1.00. Whereas two OP samples had significantly lower HSI, reported below 0.5 . All eigTo further investigate the activity of the ten unique SARS-CoV-2 probes, we analyzed their hybridization across the samples. Our data showed that five unique probes were consistently detected among all of the patient swab samples, specifically the unique probes 2, 3, 6, 7, and 9 . Of the The eight combined OP/NP patient swab samples all consistently showed hybridization to the select unique probes 2, 3, 6, 7, and 9 .Of the nine NP patient swab samples, eight showed a consistent HSI for the selected unique probes 2, 3, 6, 7, and 9 .Seven signature viral families were strongly detected amongst the swab samples from COVID patients and were consistently identified in all swab sample types, including NP, OP and combined OP/NP . Our anaAmong the NP samples from patient swabs, the respiratory agents from the viral families Bunyaviridae, Picornaviridae and Paramyxoviridae reported the strongest HSI, measuring greater than 1.0 . CoronavOn further analyses we identified, a total of 17 signatures of viral respiratory agents that were identified among patient samples and they all were detected from each swab sample type obtained . Among tWe then investigated the bacterial families that were positively detected in the patient swab samples. Our goal were to identify the bacterial families that were similar across the different samples. Twelve families were consistently identified in each of the patient sample types . Among tReviewing all the data suggests that the Steptococcaceae and Pasteurellaceae families appear to be the predominant respiratory bacterial families identified amongst the different types of samples . FurtherHaemophilus parainfluenzae was not identified in the NP samples  and Aspergillus flavus has the strongest signal intensities  maintained the strongest signal intensities  that was designed specifically to detect the SARS-CoV-2 virus, containing probes in 10 unique, 6 conserved and 3 within the regions frequently mutated. The probes are 60 nucleotides long and were designed in a way to detect different regions of the SARS-CoV-2 genome. Of the 10 unique probes, nine were designed for different regions of ORF1ab and one probe was designed for ORF8. The NSP12 (RDRP) genomic region is one of the most conserved within the coronavirus family, and so we designed three out of the six conserved probes to target this particular region.A number of reports have shown that the virus is gaining mutations that are located in specific regions of the viral genome. Based on the available mutated genomic sequences, we also designed 3 probes to detect sequences that are from these regions frequently mutates on the virus. This version of PathoChIP was also able to detect all other respiratory microorganisms that were common to the different types of samples that also contained SARS-CoV-2.6 to less than 10 reference viral RNA copies (NR-52285). We successfully detected the presence of the viral RNA genomes with precision even at dilutions of less than 10 copies. This level of specificity and sensitivity demonstrates that the PathoChip technology is a significant improvement compared to other diagnostic technologies available.We have used three different concentrations of the reference genome (NR-52285) to hybridize to probes in the unique, conserved and the mutated regions of SARS-CoV-2 on the PathoChip. All 19 probes successfully detected the SARS-CoV-2 reference genome . To deteTo further validate the effectiveness of the PathoChip for detecting SARS-CoV-2 in patient samples, we have screened NP, OP and combined NP and OP swabs, and following the same pipeline was able to detect the SARS-CoV-2 viral nucleic acids in samples from CoVID-19 patients. These results clearly demonstrated that the PathoChip technology is effective and accurate in detecting the SARS-CoV-2 nucleic acids.Streptococcous pneumoniae and Mycobacterium tuberculosis signature probes were the most and the least predominant microorganisms, respectively, that were identified in all of the patient samples. Streptococcous pneumoniae and Haemophilus influenza were two other bacterial species that maintained a high hybridization signal in the majority of samples. Similarly, Aspergillus fumigatus and Pneumocystis jirovecii are the most and the least abundant fungal species, respectively, detected all across the patient samples.There are some preliminary reports about the co-infection of different type of viruses  and bactThe PathoChIP technology provides a comprehensive tool to detect pathogenic microorganisms, including SARS-COV-2 that may be present in very low copy numbers from different types of patient samples, while also providing a detailed overview about the presence of other co-infectious agents. The presence of other bacterial and fungal respiratory agents that are common in these patients may provide additional paracrine activities that can trigger inflammatory responses and lead to differences in severity of disease associated with SARS-CoV-2 infection. Notably, the PathoChIP can detect less than 10 copies of the RNA genome for SARS-CoV-2 in a complex background of human genomic DNA, which makes it a technology with potential utility for surveillance in populations or communities where information regarding asymptomatic carriers will be important to identify and tracethus minimizing the spread of this highly transmissible agent.GSE166281).Probes for SARS-CoV-2 were selected by choosing regions of unique and conserved genomic sequences across the approximately 30,000 nucleotides which make up the entire published sequence of the virus . These sSamples for use as positive controls for SARS-CoV-2 were obtained from BEI Resources and are detailed in Following informed consent under protocols approved by the University of Pennsylvania IRB (protocol #823392), clinical specimens were obtained from 15 patients admitted to the hospital with documented COVID19 . SamplesPathoChIP is a two-channel array, with one green channel for profiling sample of interest and one red channel for reference genomic DNA (from human B cell line). Agilent Feature Extraction software was used for extraction of the raw green and red signal data from the microarray images captured by the G400DA scanner. There are spiked-in human intergenic reference probes (HRP) that were used for raw data normalization. A scale factor, which was inferred from HRP, was set as 0.3 to control the effect from red to green signal. Specifically, we multiply the log2 red signal by the scale factor to get the normalized background signal which was then subtracted from the log2 green signal to get the final normalized data. The normalized value of a probe is called hybridization signal intensity (HSI) which indicates the abundance of the species of the probe in the tested sample. All negative HSI (< 0) were set to 0 which indicates negligible signal or the absence of the species in the tested sample. HSI 0-1 means the signal intensity of a probe is 1X to 2X to the background signal. This is a positive but weak signal and indicates the low abundance of the species in the tested sample. HSI > 1 means the signal intensity of a probe is higher than 2X of the background signal. This is a positive and strong signal and indicates the high abundance of the microbial species in the tested sample. The HSI of the probes identified for specific organisms were used to create the bar plots, heatmaps and violin plots in this study. The signals from the blank swabs control arrays were used to subtract from all other arrays so that we would eliminate any potential background signals that were due to the blank swabs, VTM or other microbes that were captured in the transfer. All analyses were focused on identifying the SARS-CoV-2 probes and the common signals across the 3 samples types .GSE166281.The data presented in the study are deposited in the GEO repository, accession number The studies involving human participants were reviewed and approved by University of Pennsylvania IRB (protocol #823392). The patients/participants provided their written informed consent to participate in this study.TS, DB, and JR performed the experiments and collected the data. XL and ZW analyzed the data. DB drafted the manuscript. RC, TS, and ER revised and edited the manuscript. RC helped with the clinical samples. ER conceptualized the project. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Since the start of the pandemic until now (end of February 2021), more than 113 million people worldwide have been infected (encompassing people with and without symptoms) with SARS-CoV-2, with 89 million of those (78.8%) being classified as recovered  (2). TheEarly on, it was reported that patients after hospitalization due to SARS-CoV-2 infection may develop persistent breathlessness 53%) and cough 34%) during the following months post discharge  also complained about fatigue; another 13 and 12% patients showed signs of anxiety and depression, respectively 9% also c and prevPatients with fatigue did not show differences in autonomic functions  compared to subjects that underwent a SARS-CoV-2 infection without developing fatigue . This giIt is well-known that irritable bowel syndrome (IBS), a highly prevalent  functional gastrointestinal disorder . SymptomIn summary, a SARS-CoV-2-infection might trigger post-COVID fatigue as a variant of long COVID. Whether this is specific for SARS-CoV-2 or rather a broader risk factor for the development of a functional disease/bodily distress disorder as observed before for, e.g., post-infectious IBS, will have to be further investigated. Moreover, more emphasis should be given to the characterization of risk factors for the development of functional post-COVID symptoms so patients at higher risk can be given higher attention in a personalized approach, screened early, and provided early help to avoid over-diagnosing and iatrogenic somatic fixation. Hereby, the close collaboration of internal medicine practitioners and those specialized in psychosomatic medicine/psychiatry is key to offer treatment in an integrated manner. It is likely that treatments encouraging active participation of patients  might be more effective than passive options  as described for other functional disorders before .AS prepared the first draft of the article. All authors critically reviewed and finalized the opinion article.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.