Immunohistochemical stains are increasingly used to aid in the diagnosis of malignant melanoma, especially when the differentiation of the tumor is unclear based on examination with hematoxylin and eosin. However, aberrant expression of non-melanocytic markers has been reported in melanomas, which can sometimes be further complicated by the loss of conventional melanocytic markers. This review aims to summarize available data regarding unusual staining patterns in primary and metastatic malignant melanoma. It also raises awareness of the potential pitfalls and highlights the importance of appropriate use and interpretation of broad immunohistochemical markers in the context of clinical and histopathologic findings to facilitate the diagnosis of atypical cases of malignant melanoma. The gold standard for melanoma diagnosis is histological examination with hematoxylin and eosin (H & E). However, malignant melanoma can exhibit varied morphologic features that make definitive diagnosis challenging, as it can be mistaken for other neoplasms. Immunohistochemical stains may be employed to aid in diagnosis, especially when the differentiation of the neoplasm is unclear or when the lesion is partially sampled. In the early 1980s, S100 was first identified as a useful melanocytic marker expressing a high sensitivity approaching 100%, and it is commonly expressed in all subtypes of melanoma, including desmoplastic melanoma. A large number of other melanocytic markers have been subsequently investigated including HMB45, Melan A, tyrosinase, MITF, and SOX10, and are increasingly used in the diagnosis of melanoma ,3. NeverThe immunophenotypic profile may evolve during the course of malignant melanoma, most commonly manifesting in the loss of conventional melanocytic marker expression in metastatic lesions . In factAdditionally, Agaimy et al. reported 13 cases of metastatic malignant melanoma with complete loss of differentiation markers. All cases were negative for S100, HMB45, Melan A, and SOX10. Aberrant expression for smooth muscle actin (SMA), pancytokeratin, and desmin was also noted. The diagnosis was made after exploration of the remote history and with the help of molecular testing [It is well known that desmoplastic melanoma often stains negative for specific melanocytic markers such as Tyrosinase, HMB-45, and Melan A . S100 prShinohara et al. reported an unusual case of primary melanoma from the right temple of a 62-year-old man with loss of staining for S100 protein, HMB-45, and Melan A, but expressed tyrosinase. Interestingly, regional lymph node metastases showed positive S100 protein, MITF, and tyrosinase staining, further emphasizing the protean nature of melanoma. The morphologic features of the tumor were of a poorly differentiated carcinoma, melanoma, or other neoplasms. Additional markers for carcinoma, vascular, and hematopoietic neoplasms were all negative. This case highlights the fact that, although tyrosinase is reported to be less sensitive than S100 protein, it is one of the most specific immunostains that may prove useful when the diagnosis of malignant melanoma is in doubt . This caChang and Argenyi reported another case of primary melanoma on the back of a 58-year-old man, in which the central portion of the tumor had lost expression of common melanocytic markers and expressed aberrant epithelial markers including pancytokeratin and CMA 5.2. Sentinel lymph node biopsy showed a similar cytomorphology to the primary tumor, with loss of expression of melanocytic markers, in addition to aberrant staining with PAX-8 and the epithelial markers mentioned previously. Several months later, the patient presented with a subcutaneous nodule in his right lateral hip. The excision revealed a malignant neoplasm with positive reactivity to pancytokeratin, CAM 5.2, and PAX-8, in addition to S100, SOX10, and Melan-A. Renal cell carcinoma was suspected based on PAX-8 immunoreactivity. However, no primary renal tumor was found on imaging studies. Given the similar immunophenotype as the primary melanoma and immunohistochemical evidence of melanocytic differentiation, the lesion was interpreted to be a metastatic melanoma .The aberrant expression of desmin in melanoma was initially reported in a single case by Truong et al. . Later oOsteogenic melanoma with aberrant expression of desmin was reported in a 49-year-old woman who presented with a pigmented lesion in the subungual region of her left hallux. The tumor was composed of pleomorphic atypical epithelioid and fusiform cells with focal lentiginous proliferation of large, atypical melanocytes along the dermoepidermal junction. There were also areas of osteoid matrix that were focally mineralized. Immunohistochemistry revealed expression of S100 protein and desmin. Further, 40% of the neoplastic cells were weakly positive for Melan-A, HMB45, and MITF. The patient underwent amputation of the left hallux with negative sentinel lymph node . In one On the contrary, Banerjee and Harris reported that α-SMA positivity is extremely uncommon in conventional cutaneous or metastatic melanomas . HoweverAdditionally, one study has shown that malignant melanoma cells release a platelet derived growth factor-like substance that inhibits the expression of smooth muscle alpha-actin and has a suppressive effect on the contractile elements in non-neoplastic cells. The study has found that, in close vicinity to the tumor, the SMA in the vessels walls was often discontinuous and obscure, and no melanoma cells were stained with SMA. These results suggest that tumor cells can release factors that alter the cytoskeletal system in the surrounding stroma .Calponin is a 32 to 36 kDa protein that is associated with the cytoskeletal fraction of actin, and is identified in smooth muscle cells, myofibroblasts, and myoepithelial cells. Lee et al. reported three cases of sinonasal melanoma focally positive for Calponin. In two cases, the cells with calponin immunoreactivity appeared to be the same cells stained with chromogranin. None of these cases showed spindle cell morphology or desmoplastic reaction .Aberrant expression of neurofilament protein and glial fibrillary acidic protein has only very rarely been reported in malignant melanoma. Romano et al. investigated the expression of these markers in 71 patients diagnosed with malignant melanoma. Here, 5/31 (16%), 3/32 (9%), and 10/34 (29%) of the cases were aberrantly positive for neurofilament protein, glial fibrillary acidic protein, and synaptophysin, respectively, confirming the prior published observations. In contrast, chromogranin A expression, which usually parallels that of synaptophysin, was absent in all cases . AdditioCD56, also known as neural cell adhesion molecule, is normally expressed on neurons, skeletal muscle, and natural killer cells. However, it lacks the specificity of the neuroendocrine markers mentioned above. Aberrant CD56 expression in primary esophageal melanoma was reported in one case. The neoplastic cells also stained positive for S100, Melan A, and SOX10 and negative for cytokeratin Synaptophysin, Chromogranin, p63, TTF-1, LCA, and other lymphoid markers. A primary esophageal melanoma can cause diagnostic difficulty, particularly when a small biopsy is available for evaluation . In factAdditionally, in a case series of 14 patients published by Steppert et al., CD56 was positive in about half of the samples. For primary melanoma, CD56 was weakly positive in 2/4 cases. For the metastases, 20 specimens were evaluated. CD56 was strongly positive in 4 cases, weakly positive in 5 cases, and negative in 11 cases. Further, 8/15 specimens also stained positive for synaptophysin. No specimen stained for Chromogranin A. Single cell positivity can be found in CD56-positive tumor infiltrating lymphocytes, but the strong positivity is considered relevant when the tumor cells themselves express CD56 .Keratin in melanoma was first reported by Gatter et al. The authors noted immunoreactivity with CAM 5.2 antibody in 4 of 41 fresh frozen melanomas. However, they did not find keratin expression in routinely processed melanomas . These rPlotzke et al. reported the expression of CK cocktail in 1.2% (3/248) of melanoma cases, only one of which was diffusely positive. All three cases were epithelioid metastatic melanomas without spindle cytomorphology. The association between cytokeratin cocktail positivity and metastasis was statistically significant .Furthermore, Saggini et al. reported a case of primary intrafascial desmoplastic melanoma with aberrant cytokeratin AE1/AE3 expression, arising on the scalp of a 71-year-old man. Histologically, the tumor exhibited a biphasic pattern, with a predominating hypocellular proliferation of spindled cells and foci of polygonal to round epithelioid cells. On immunohistochemistry, both populations were diffusely positive for S100 and SOX10 and negative for HMB-45, CAM5.2, CK-7, CK-20, EMA, SMA, desmin, GFAP, TTF-1, CDX-2, and p63. Additionally, the epithelioid, but not the conventional desmoplastic part, exhibited strong staining with pan-cytokeratin AE1/AE3 .In summary, the expression of epithelial markers in melanoma is not uncommon, and it appears to be more frequent in cases with epithelioid cytomorphology . HoweverAberrant CD68 expression was reported in one case of primary amelanotic melanoma in ascites fluid of a 67-year-old man. The strong expression of CD68 and the absence of brown pigment were unusual. Negativity for epithelial markers including CK5/6, CK7, CK20, p63, CDX2, TTF1, PSA, and GATA3 helped to exclude the possibility of a carcinoma. Tumor cells were found positive for S100 and negative for non-epithelial markers including CD3, CD20, CD30, MUM1, and HHV8. In order to distinguish fibrohistiocytic tumor from malignant melanoma, immunostaining for CD163 and clusterin, together with melanocytic markers, was used. Strong nuclear SOX10 expression and a moderate reaction for Melan A were observed. Negative immunostaining for CD163 and langerin was also helpful to exclude histiocytic lymphoma and Langerhans cell Histiocytosis . In summAnother case of malignant melanoma with loss of conventional melanocytic markers except Tyrosinase showed immunostaining in both the primary lesion and lymph node metastasis . CD68 imShah et al. reported that CD68 is consistently expressed in primary conventional, desmoplastic, and metastatic melanomas, but negative for neural tumors and for dermal component of compound nevi, making it a useful marker for the diagnosis and differential diagnosis of such neoplasms .Jensen et al. reported expression of CD163 in 35% and CD68 in 10% of 190 melanomas. It is interesting to note the CD68 expression was independently associated with poor relapse-free survival and CD163 expression was an independent prognostic factor along with tumor thickness [The induction of an adequate tumor vasculature in response to the increasing demand of oxygen is important for tumor growth and invasion. Melanoma cells are thought to induce vasculogenic mimicry, which refers to the tumor cell’s ability to express endothelium-associated genes and form a vasculogenic-like patterned network . AberranPisacane et al. demonstrated the immunohistochemical expression of CD31 and CD34 in 22/30 (73%) and 10/30 (33.3%) of invasive cutaneous melanomas cells, respectively. CD31, but not CD34 also stained 10 of 15 metastatic melanomas. Additionally, CD31 and CD34 expression was never found in common melanocytic nevi. The authors also found that CD31 and CD34 expression closely parallels the morphologic phases of melanocytic tumor progression corresponding to Clark’s levels and their associated metastatic potential .Salven et al. also reported that VEGF is upregulated during the course of melanoma progression. The study showed that lesional cells are significantly more likely to stain for VEGF in metastatic versus primary melanomas . Tumor infiltrating inflammatory cells also expressed VEGF in all melanomas .CD34 has been described as a marker of dermatofibrosarcoma protuberans (DFSPs). Its expression has also been documented in benign neural tumors including neurofibroma and schwannoma, and spindle cell neoplasms such as solitary fibrous tumors and giant cell fibroblastoma . CD34 exExpression of hematopoietic markers in non-hematopoietic neoplasms is extremely uncommon. Chatzopoulos et al. observed a strong tumor cell membranous reactivity for CD4 in two cases of metastatic melanoma. The authors considered the hypothesis of CD4+ lymphocytes phagocytosis by melanoma tumor cells with resulting immunoreactivity or even the possibility of an artifactual cross-reactivity due to endogenous Fc receptor expression by tumor cells. The exact significance and mechanism of CD4 expression are unknown and remain to be elucidated by further studies .Fang et al. identified a small subpopulation of melanoma cells with stem cell-like properties that express CD20, but are negative for CD3, CD4, CD8, and CD45. Furthermore, these cells were more tumorigenic than adjacent cells when grafted to mice. It is possible that this aberrant expression of hematopoietic cells may allow melanoma cells to easily access the circulation and establish distant metastases .FLI-1 nuclear transcription factor has been proposed as a tool in the differential diagnosis of small round cell sarcomas. It has also been described as a marker in endothelial differentiation. Expression of FLI-1 has been demonstrated in Ewing’s sarcoma/primitive neuroendocrine tumor, small cell carcinomas of the lung, and benign and malignant vascular neoplasms. Strong expression of FLI-1 has been reported only in seven cases of melanoma that displayed variable expression of melanocytic markers . OverallGATA3 is commonly used as a marker for mammary and urothelial carcinoma, especially in the setting of distinguishing the origin of metastatic carcinoma. However, it is not entirely specific as it can also be expressed in skin tumors including both epidermal and adnexal neoplasms, but not in melanoma . MetastaCEA immunoreactivity was shown to be expressed in 15 of 28 cases of metastatic melanoma when polyclonal antibodies were used. No staining was seen with monoclonal anti-CEA antibodies . A studyAnother study reported all 12 cases of primary melanoma to express polyclonal CEA, while only 1 of 11 Spitz nevi was positive for this marker, highlighting the potential for this antibody to distinguish between these two entities .Calretinin is a calcium binding protein that is structurally related to S100 protein and inhibin. Immunohistochemical stain for calretinin shows nuclear and cytoplasmic staining in mesothelial cells, adipocytes, endometrial stromal cells, ganglion cells, Sertoli cells, and Leydig cells. Aberrant staining for calretinin stain is rare in melanoma . MetaplaMetaplastic melanoma has not been previously reported to stain for calretinin. Rare incidents of calretinin positive melanoma are reported in epithelioid, pleomorphic, and metastatic melanoma. The awareness of the possibility of aberrant calretinin positivity in metaplastic melanoma with chondroid differentiation is critical to avoid a potential pitfall in misdiagnosing metaplastic melanoma as sarcoma or mesothelioma .PAX8 and PAX2 are common markers for renal or Mullerian differentiation. While most PAX8+ and PAX2+ carcinomas are rarely confused with melanoma, cases of MiTf family altered renal cell carcinoma (MiTF-RCC) may pose a diagnostic challenge owing to the frequent patchy expression of melanocytic markers. In a study of 263 melanomas, the expression of PAX8 was reported in 7.9% of cases and was significantly associated with spindle cytomorphology. PAX2 was positive in one (0.4%) case. In summary, caution should be exercised when evaluating a skin tumor in patients with history of MiTF-RCC as a small subset of primary or metastatic melanomas may demonstrate PAX8 or PAX2 staining. Ultimately, these markers should be used in a broad immunohistochemical panel rather than in isolation, in addition to molecular or cytogenetic studies when in doubt .In conclusion, malignant melanoma showing aberrant expression of immunohistochemical markers may easily be mistaken for carcinomas, lymphomas, sarcomas, and neuroendocrine tumors. Luckily, the majority of these cases showing aberrant expression will stain positive for at least one specific melanocytic marker. Awareness by pathologists of these potential pitfalls, and appropriate use and interpretation of broad immunohistochemical markers together with the clinical history, should facilitate the diagnosis of malignant melanoma with unusual staining patterns.


The fast, reliable, and accurate identification of IDPRs is essential, as in recent years it has come to be recognized more and more that IDPRs have a wide impact on many important physiological processes, such as molecular recognition and molecular assembly, the regulation of transcription and translation, protein phosphorylation, cellular signal transduction, etc. For the sake of cost-effectiveness, it is imperative to develop computational approaches for identifying IDPRs. In this study, a deep neural structure where a variant VGG19 is situated between two MLP networks is developed for identifying IDPRs. Furthermore, for the first time, three novel sequence features—i.e., persistent entropy and the probabilities associated with two and three consecutive amino acids of the protein sequence—are introduced for identifying IDPRs. The simulation results show that our neural structure either performs considerably better than other known methods or, when relying on a much smaller training set, attains a similar performance. Our deep neural structure, which exploits the VGG19 structure, is effective for identifying IDPRs. Furthermore, three novel sequence features—i.e., the persistent entropy and the probabilities associated with two and three consecutive amino acids of the protein sequence—could be used as valuable sequence features in the further development of identifying IDPRs. Protein regions which lack stable three-dimensional structures are referred to as intrinsically disordered regions (IDPRs) . In receThere are many computational methods for identifying IDPRs. These methods can be divided into three categories: (1) Physicochemical-based methods, such as FoldIndex , GlobPloWhile all of the above methods have contributed to the development of the field, there are still some new features that have not been discovered. Because of the interaction between amino acids, the question of how to describe them is key to improving predictions based on protein sequences.In this paper, we develop a deep neural structure composed of a variant VGG19 , where tIn this section, the datasets used in this paper for training and blind testing are presented. The features extracted from the training dataset are depicted. In particular, we introduce three novel features, which are used for the first time for identifying IDPRs. These three novel features are persistent entropy based on persistent homology, PCAA2, and PCAA3.The dataset DIS1616 from the DisProt  ,),…,H2(L)H3=H3(1),In view of  and 8),,8), funcI3(j) in  and 12)I2(j) andN3(·) in  and (12)In  and 18)18), we hIn this section, we illustrate how to compute the input of our deep neural network, which is composed of 35 features derived from a protein sequence. Of these 35 features, there are twenty evolutionary features which are determined through the PSSM  computedL, we choose a sliding window of odd size NN consecutive amino acids. Then, for these amino acids in the sliding window, we compute the evolutionary features, physiochemical properties, and propensities, as defined in the previous paragraph. These thirty computed feature values of amino acids in the sliding window are averaged and the averaged results are used to represent the feature values of the amino acid in the center of the sliding window. For simplicity, we first transform N, we also compute the Shannon and topological entropy through the procedure from Equations (1)–(14), as described in the paper withxj=[vjm34In this section, we develop a deep neural structure composed of a variant VGG19, where the variant VGG19 is situated between two MLP networks used for identifying IDPRs. Then, we introduce the process of training the deep neural network.The overall architecture of our model, as shown in fined in  as its ii is the i-th amino acid in the protein sequence The output of the variant VGG19 is a lgorithm  with a dm  is therefore defined as:The total loss function of our model for a package of size i-th amino acid is disordered, or to 0, implying that it is ordered.In Equation , the preIn this section, we present the process of training the deep neural network developed in the previous section. The training dataset we use in this paper is DIS1450 from the DisProt . We put Four metrics were used to evaluate the performance of IDPR prediction . These wWe use http://bliulab.net/RFPR-IDP/server (accessed on 26 March 2021)), SPOT-Disorder2 (available at https://sparks-lab.org/server/spot-disorder2/ (accessed on 26 March 2021)), DISvgg [In this section, we will demonstrate the performance of our deep neural network on the different test sets: DIS166 , R80 2525, and M, DISvgg . For conOn the test sets DIS166, R80, and MXD494, the performance of MLP-VGG19-MLP was superior to that of RFPR-IDP, SPOT-Disorder2, and DISvgg. The  used in , attains used in . Table 2In this study, a deep neural structure is developed for identifying IDPRs, where a variant VGG19 is situated between two MLP networks. Furthermore, for the first time, three novel sequence features—i.e., persistent entropy, PCAA2, and PCAA3—are introduced for identifying IDPRs. In comparison with our previous DISvgg algorithm, the prediction performance of MLP-VGG19-MLP exceeded it. Furthermore, only one VGG19 was used in this paper, while ten VGG16nets were employed in the previous paper. In comparison with RFPR-IDP, SPOT-Disorder2, and IDP-Seq2Seq, MLP-VGG19-MLP relies on a much smaller training set to achieve a performance that is better or similar to that achieved using other methods. The simulation results show that our neural structure either considerably outperforms other known methods or, when relying on a much smaller training set, attains a similar performance. Three novel sequence features could be used as valuable sequence features in the further development of identifying IDPRs.


A lack of knowledge and inadequate practices of health care providers (HCPs) are the main obstacles to effective cancer pain management (CPM). The main objective of the study was to evaluate the CPM knowledge, CPM practice, and attitudes towards pharmacists' participation and advanced methods in CPM of physicians, nurses, and pharmacists in China.  An open online survey was adopted using social media software (WeChat) as the platform to conduct a nationwide survey of HCPs involved in CPM in public medical institutions at all levels in China from March to June 2019. ptrend <0.05).  A total of 1279 physicians, 2267 nurses, and 1466 pharmacists participated in the survey. Among the three types of professionals, nurses had the highest level of practical ability (61.63 ± 28.99) and best attitudes towards pharmacists' participation and advanced methods in CPM (72.05 ± 33.71) and physicians had the best mastery of CPM-related knowledge (69.60 ± 28.45), while pharmacists performed the worst in these three aspects . Only 19.69% of the hospitals had a pharmacist to tumor patient ratio ≥1 : 50. Hierarchical analysis showed that passing a good pain management (GPM) ward program and participating in advanced training had positive impacts on the scores of all three parts in the three professions ( HCPs' levels of practice, knowledge, and attitudes towards pharmacists and advanced methods of CPM were average in China; however, pharmacists had the worst performance, which demonstrates a need for further improvement. Furthermore, GPM ward programs and advanced trainings are helpful for improving CPM levels. Pain is highly prevalent among patients with cancer, especially those with advanced or metastatic cancer . AdequatCancer pain can be effectively reduced through appropriate drug treatment ; howeverBased on all the above information, this study involved the design of a set of questionnaires to simultaneously evaluate the knowledge and practice regarding CPM of physicians, pharmacists, and nurses at different levels of public hospitals and departments in China. Unlike the existing studies with pharmacists –19, thisAs China has started to promote the WHO “3-step analgesic ladder” Program and launch the good pain management (GPM) ward program, some progress has been achieved in CPM . Neverthhttp://www.wjx.cn/), and only after all questions were completed online as required could the questionnaire be submitted. Therefore, any responding questionnaire successfully received by the system was valid. As an open online survey, the participants voluntarily participated, potentially leading to nonrepresentativeness of the respondents. In this study, an overrepresentation of motivated respondents with experience of, and interest in, CPM were considered to be supportive of, rather than disabling to, our aims.This cross-sectional online survey was conducted in public medical institutions at all levels in China from March to June 2019. All HCPs from the Departments of Medical Oncology, Surgical Oncology, Pain, Anesthesiology, etc. involved in CPM were eligible for participation. The survey utilized the Blue Ribbon Cancer Pain Patients Care Cooperation Group as the promoter, recruiting volunteers through the WeChat online social media platform or via face-to-face communication in the palliative care training sessions. Furthermore, the survey invited 1 national well-known clinical leader and 14 local well-known experts in this field to circulate the questionnaire via their networks, to effectively distribute it throughout the country. All participation in the survey was voluntary and anonymous. Approximately 10–15 minutes was required to complete the questionnaire on the online platform Questionnaire Star  to assess wording, structure, layout, and readability and to provide an estimate for completion time. The pilot study resulted in very minor modifications to the word expression and data from the pilot participants were not included in the final analysis.The questionnaire was created by researchers according to the purpose and requirements of the survey and revised by a panel of experts comprising medical oncologist (S. L. Y.), pharmacologists (Y. Z. and C. S.), nurse (C. F. P.), palliative care expert (J. L. H.), and epidemiologist (L. L. Y.). The piloted survey was carried out in one hospital by a small number of HCPs  basic information; (2) CPM practical ability; (3) knowledge about CPM; and (4) attitudes towards pharmacists' participation and advanced methods in CPM. The first section consisted of 9 questions on background characteristics of the sample including occupation, professional title, religion, hospital grade, and departments. The second section contained 6 questions for assessing the practice of HCPs in CPM from the follow-up frequency of discharged cancer pain patients, the use of pain assessment tools, the timing of pain assessment, the notification of adverse reactions, and the choice of analgesics. In the third section, knowledge of CPM was evaluated through 6 items , tapping into the mastery of the guidelines, opioid dose calculation, the specific properties of analgesics, and the principles of CPM. The final section consisted of 9 questions  to assess HCPs' attitudes towards pharmacists and advanced CPM methods. Participants were asked to indicate whether there were pharmacists participating in CPM in their hospital, in what forms, and their needs for pharmacists and advanced CPM methods in their work.α for the questionnaire was 0.89, 0.84, and 0.83 for physicians, nurses, and pharmacists, respectively, indicating acceptable internal consistency.Several questions in the questionnaire were answered by specific occupational groups. Question 6 (“What proportion of patients under your care had cancer-related pain?”) was for doctors and nurses only, while questions 14  and 15 (“What are the reasons why you prefer to use these drugs?”) were for doctors only. Cronbach's In addition to the 9 basic information items, 7 of the remaining 21 items were used for descriptive analysis  × prenormalized scoreThe equation used to calculate the normalized score for multiple-choice questions  is normaTo facilitate analysis, all scores were normalized to a 0–100 numeric rating scale.The overall score of the questionnaire was the average normalized score for the all the 14 items, and the aspect  score was the average score of all of the items in each aspect. Both the total score and aspect score ranged from 0 to 100 points. The evaluation criteria for the score were as follows: <50 points was considered “poor,” 50–75 points was considered “normal,” and ≥75 points was considered “good” .P < 0.05 were considered statistically significant.Differences in the scores of physicians, nurses, and pharmacists were tested by using the Kruskal–Wallis nonparametric test. Binary logistic regression analysis was used to evaluate the associations between different factors and HCP scores >65 points. In this model, “score” was classified as a dummy variable . Sixty-five points was used as a cutoff value for stratification, as the median overall score of HCPs was 63.47 points. Stratified analyses were performed to analyze the influence of six major characteristics of the study population , passing of the GPM ward program, number of CPM trainings received per year, and types of CPM trainings) on the scores of participants in different occupations. All statistical analyses were performed using IBM SPSS Statistics 23.0. Results with A total of 5012 participants were investigated in this study, with 1279 doctors, 2267 nurses, and 1466 pharmacists. The proportion of HCPs from grades 3 and 2 and lower hospitals was 75.12%, 20.39%, and 4.49%, respectively. In China, grades 3, 2, and 1 hospitals are responsible for approximately 80%, 15%, and 5% of the diagnosis and treatment of patients with cancer, respectively [P < 0.01) , followed by physicians (57.00 ± 28.74) and pharmacists (50.04 ± 26.69) ( < 0.01) . It can  < 0.01) .The analgesic drugs most commonly used by physicians in this survey were oxycodone hydrochloride sustained-release tablets (53.24%), morphine sustained-release tablets (45.82%), morphine immediate-release tablets (40.50%), morphine injections (36.36%), and fentanyl transdermal patches (18.45%). Additionally, the usage rate of pethidine hydrochloride (dolantin) ranked sixth, at 9.46%. The main factors that determined the analgesic drugs commonly prescribed by physicians included the following: better analgesic effect (60.99%), ease of use and acceptance by the patients (53.79%), lower incidence of adverse reactions (45.43%), higher safety (45.19%), and guideline recommendations (39.72%)   , which w < 0.01) .Regarding the functional requirements of the management system, HCPs had the highest requirements for six functions: automatic screening; identifying pain patients; online pain assessments; intelligent delivery of pain orders; online conversion of different opioid dosages; online communication between physicians, pharmacists, and nurses; and regular delivery of popular science articles (Supplementary ptrend <0.05). However, the remaining three factors had heterogeneous effects on the scores on the three parts.Stratified analyses were subsequently conducted to analyze the influence of six major characteristics of the study population  on the sP < 0.01), respectively (P < 0.01)) ) , all of P < 0.01) (P < 0.01)  were significantly higher than those of physicians and nurses  , which w < 0.01) , indicat < 0.01) . Clinici < 0.01) . In CPM,ptrend<0.05). Since 2011, China has started to engage in the GPM ward program nationwide to improve the management of cancer pain in China. This study confirmed for the first time that the creation of a GPM ward was very beneficial for increasing the practice, knowledge, and attitude of HCPs [According to the stratified analysis of different occupational groups, professional title, hospital level, proportion of patient load accounted for by cancer pain patients, GPM ward evaluation status, number of annual trainings, and diversity of forms of training had different effects on the scores of the respondents. Presently, the literature on CPM practice, knowledge cognition, and attitude of physicians, pharmacists, and nurses is insufficient. Most of the established reports believe that the CPM knowledge of physicians or pharmacists is better than that of nurses, and nurses are better than physicians or pharmacists in pain assessment , 25, 31. of HCPs . The eff of HCPs . Several of HCPs , 35–37,  of HCPs , 38, 39. of HCPs ,41, indi of HCPs , 32, 42. of HCPs , the per of HCPs , 30, 43. of HCPs , 45. WheWhen considering the results of this study, some important limitations must be taken into account. First of all, most of the CPM knowledge questions were subjective, and no comprehensive case questions  were used to test the true knowledge level of the HCPs. Second, although the options of hospital level and geographical location were set in the questionnaire, there was no requirement for participants to fill their working hospitals, so it was impossible to know how many hospitals were covered in this survey. Third, the scores of different types of questions had been corrected, which may have had an impact on the statistical analysis. Finally, the setting of the questions was not comprehensive, which hindered further in-depth assessment and analysis.Although there were some defects in this survey, as a national cross-sectional survey, the sample size was up to 5012. These subjects came from all over the country and were evenly distributed in different levels of hospitals. Moreover, our findings were highly consistent with those of previous survey studies from China and abroad. Therefore, this survey could truly reflect the current situation of cancer pain management in China to a certain extent and provide reference for the improvement of cancer pain management.In conclusion, HCPs' levels of practice and knowledge of CPM were average in China; however, pharmacists had the worst performance. The attitudes towards pharmacists' participation of HCPs showed that there is a need for pharmacists to participate in CPM, but faced with the short supply of pharmacists. Therefore, China urgently needs to strengthen the education and development of more competent clinical pharmacists to fully participate in the multidisciplinary management of cancer pain. In addition, we can try various training methods to enhance the quality and effect of HCP training, encourage hospitals to create GPM wards, and introduce advanced CPM methods to improve CPM success.


To estimate the prevalence of clinical signs and symptoms of severe/extreme stress, anxiety, and depression, as well as their associated factors, among Brazilians during social distancing. This is a cross-sectional study conducted in April/May 2020 with 3,200 Brazilians over 18 years old. Respondents’ sociodemographic and clinical data were collected using an online questionnaire, which also included the 21-item Depression, Anxiety and Stress Scale (DASS-21) to assess emotional symptoms. Unadjusted and adjusted prevalence ratios and their respective 95% confidence intervals were estimated using Poisson regression models with robust variance. Our results show the prevalence of severe/extreme stress was 21.5%, anxiety 19.4%, and depression 21.5%. In the final model, sociodemographic, clinical, and Covid-19-related factors were associated with severe/extreme stress, anxiety, and depression in Brazilians during social distancing due to the Covid-19 pandemic. We found the main factors associated with severe/extreme depression to be young women, brown, single, not religious, sedentary, presenting reduced leisure activities, history of anxiety and depression, increased medication use, and Covid-19 symptoms. This study may help develop and systematically plan measures aimed to prevent, early identify, and properly manage clinical signs and symptoms of stress, anxiety, and depression during the Covid-19 pandemic. Among the recommendations to prevent severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spread1, social distancing has proved to be effective in containing the disease2.Emerging infectious disease in humans may require preventive measures to contain infections spread. However, these measures tend to influence individuals’ behavior and interaction with each other1. Mental well-being plays a seminal role in the overall health, so that mental disorders incur functional disabilities, reducing individuals’ quality of life, increasing healthcare-related costs, and damaging interpersonal relationships3.Studies have shown that social distancing affects mental health, constituting a risk factor for mental disorders4, be it direct or indirect. Directly, the pandemic affects individuals’ mental health through the virus neurotropic properties, and indirectly through excessive concern over one’s future during social distancing5. However, studies in different countries still need to be consolidated for providing more information on the subject.Recent evidence suggests the Covid-19 pandemic has a psychological impact on individuals6. However, the literature suggests that Covid-19 crisis increased these numbers, as the country recorded a higher prevalence of depression (61.3%), anxiety (44.2%), stress (50.8%), and psychological impact (54.9%) during the pandemic7. Likewise, China, the first country to adopt social distancing measures against the virus spread, reported rates of psychological distress ranging from 7% to 53.8% during the initial stage of the Covid-194.Before the pandemic, the prevalence of anxiety disorders was 9.3% in Brazil, whereas for depression this index was 5.4%, ranging from mild to extreme8.According to studies conducted during social distancing, the main risk factors for stress, anxiety, and depression are being female, a healthcare worker, over 50 years old, having poor education level, presenting some type of physical morbidity or psychiatric disorder, and having a friend or relative with Covid-19 symptoms12, our results should be considered as a whole for planning and developing health actions aimed at this population. Moreover, we explored risk factors not previously addressed in the literature, be it national12 or international8.Considering the importance of data availability in preventing mental disorders, the exponential increase in the number of cases and deaths in Brazil, and the implemented social distancing measures, this study evaluated the prevalence of severe/extreme stress, anxiety, and depression in the Brazilian population and determined the factors associated with these symptoms during the Covid-19 pandemic. Added to other studies on the same themeThis is a prospective cross-sectional study conducted in April and May 2020. Native or naturalized Brazilians living in Brazil or abroad were eligible for inclusion. Individuals who failed completing the questionnaire and those under 18 years old were excluded.13.First, all investigators involved in the study sent the questionnaire to their contacts via Instagram, Facebook, WhatsApp, and Twitter, and then asked those to forward the survey to all their contacts on social media, advertising the research.To collect participants’ sociodemographic and clinical data, an online questionnaire with easily understandable objective-response questions was developed using the Google Forms platform, including the 21-item Depression, Anxiety and Stress Scale (DASS-21) validated for BrazilIndependent variables consisted of participants’ sociodemographic data, such as age, gender, race/ethnicity, education level, marital status, geographic region of residence, individuals per household, number of children, rooms per household, and religion.Participants also reported their activities – including the practice of leisure activities, physical exercise, and Internet usage before and during social distancing – and whether they have a medical history of anxiety and depression, chronic diseases, illicit drug use, smoking, alcohol consumption, and medication use. Regarding work-related activities, participants were asked about their occupation, employment status, monthly income before and after social distancing measures implementation, and whether they were able to work from home.14.Based on a previous research, this study nominally categorized the variables practice leisure activities, physical exercise, internet usage, smoking, alcohol consumption, medication use, and working from home during social distancing as “increased,” “reduced,” “I do not perform/use,” or “remains the same”1, as well as social distancing duration. The questionnaire also contained disease-related questions, such as whether any family member or acquaintance have been infected with the disease, whether they had contact with someone suspected or confirmed to have it, and whether themselves presented symptoms, as well as any test results on the disease.Respondents also reported the preventive measures adopted by them aimed at preventing SARS-CoV-2, such as quarantine, social distancing, or social isolation13.Dependent variables consisted of clinical signs and symptoms of stress, anxiety, and depression, according to each subscale of DASS-21, used to identify emotional symptoms. Based on a 4-point Likert-type scale, DASS-21 required participants to report the degree to which they had experienced each symptom (described in statements) over the preceding week. In the stress subscale, scores ranging from 0–14 indicate normal, from 15–18 mild symptoms, from 19–25 moderate, from 26–33 severe symptoms, and > 33 indicate extremely severe symptoms. As for the anxiety subscale, scores ranging from 0–7 are classified as indicative of normal, 8–9 mild, 10–14 moderate symptoms, 15–19 severe, and > 19 extremely severe symptoms. Finally, in the depression subscale, scores ranging from 0–9 are indicative of normal results, 10–13 mild symptoms, 14–20 moderate, 21–27 severe, and > 27 extremely severe symptoms9  and considering a 80% power and a 95% significance level, 810 patients will be necessary for the study.Sample size was determined using the Epi Info StatCalc software version 3.5.1 . Predicting a 5% rate of depression among BraziliansAll analyses were performed on Stata version 12.1 . First, a univariable analysis was conducted to describe the dependent and independent variables distribution in the study population. Unadjusted and adjusted prevalence ratio (PR) and their respective 95% confidence intervals (95%CI) were calculated using Poisson regression with robust variance. The significance of each explanatory variable present in the model was evaluated using the Wald test. In the multivariable analysis, the initial model consisted of the explanatory variables with p < 0.20 in the univariable analysis; variables with significance level < 0.05 remained in the final model.This study was approved by the institutional review board of the Universidade Católica de Pernambuco, under reference number 3.988.875 on April 24, 2020 and CAAE 30623020.1.0000.5206. All participants received an online informed consent form and checked either “sign” or “do not sign,” so that only those who agreed to participate and chose to sign were included in the study.Of the 3,793 individuals who responded the questionnaire, 3,200 were included in the study (84.4%). We excluded the forms of 565 respondents (14.9%) who failed in fully completing the questionnaire or who answered it in duplicate, and of 28 (0.7%) who were underage.Regarding participants’ epidemiological characteristics, we verified that 21.3% (n = 681) were aged between 34 and 44 years, 45.2% (n = 1446) were catholic, and 7.0% (n = 223) reported to have been socially distancing for 10 days or less. Most respondents were women , white , single , undergraduate students , and lived in the North-Eastern region of Brazil  .Based on symptoms severity, we found the prevalence rate of stress within the study population to be equal to 21.5%, of anxiety to be qual to 19.4%, and depression to 21.5% .In the final model, risk factors associated with clinical signs and symptoms of severe/extreme stress were: age between 18 and 20 years ; being of female gender ; living abroad ; having a history of anxiety and depression ; having reduced leisure activity practice ; having increased medication use ; and complying with social isolating measures . Conversely, being able to work from home  and using the Internet for leisure activities  were protective factors against stress ; being brown-skinned ; having reduced possibility of working from home ; living in the North region of the country ; having no more than 1–6 rooms within the household ; having a history of anxiety and depression ; having a chronic disease ; having reduced leisure activity practice ; having increased medication use ; and complying with social isolating measures  or quarantine . Protection against severe/extreme anxiety increased alongside age, specially within the 45-83-year age group  ; being of female gender ; being brown-skinned ; being single ; presenting decreased family income ; having reduced possibility of working from home ; being unemployed ; living abroad ; having no more than 1–6 rooms within the household ; not being religious ; having a history of anxiety and depression ; having reduced leisure activity practice ; having reduced physical activity practice ; reduced or null Internet usage ; having increased medication use ; using illicit drug ; complying with social isolation measures ; and presenting Covid-19 symptoms  , anxiety (44.2%), stress (50.8%), and psychological impact (54.9%)7, similar to those found in this study when combining mild, moderate, and severe cases. These differences may stem from varying contexts between countries, distinct regions within the same country, or from different methodologies and approaches to measure clinical signs and symptoms. Our research was conducted during a period of exponential increase in the number of cases and deaths in Brazil (ranking 4th worldwide regarding the number of cases), with more than 240 thousand cases and 16 thousand deaths21.The prevalence rates of signs and symptoms of severe/extreme stress, anxiety, and depression were 21.5%, 19.4% and 21.5%, respectively. These rates corroborate a Spanish study that reported prevalence rates of 18.7% for depression and 21.6% for anxiety, without classifying the disorders according to severity22. Our study, on the other hand, verified a greater prevalence of mental disorders among younger individuals (under 45 years old) and undergraduate students . We also found older age to function as a protective factor against mental disorders. A study conducted in a similar setting also reported a greater psychological impact on younger individuals23, which may be explained by the lower maturity level among this population and their fewer resources to deal with social distancing.Studies suggest that older individuals have greater risk of morbidity and mortality related to Covid-19, besides being more prone to mental disorders 12 and other countries, which report a greater risk among women in similar social distancing conditions24. This might be explained by women’s socially imposed role as family caregivers, making them assume household chores besides their occupation, which overburden them physically and psychologically and increase their susceptibility to mental disorders20. A study conducted prior to the pandemic already demonstrated that women were more likely to develop psychological disorders, either as a result of their double workload and/or hormonal influences16.We found women to be more likely to suffer from severe/extreme stress, anxiety, and depression. This finding is corroborated by studies conducted in Brazil25. According to the authors, this may be explained by a history of discrimination and its enduring consequences, which are social factors that increase the risk of psychological symptoms among those “non-white”25.Although individuals’ skin color/ethnicity tend to affect their educational, economic, and social opportunities, no consensus has been reached regarding mental disorders association with different ethnic groups and labels. We found self-reported brown skin to constitute a risk factor for severe/extreme anxiety and depression. A systematic review conducted in Brazil, in which race was dichotomized into “white” or “non-white,” found “non-white” individuals to be associated with mental disorders23 – an important factor, given that this group already presents a high prevalence rate for psychological disorders in ordinary situations27.Besides the social distancing measures imposed by Covid-19 pandemic, undergraduate students also had to face the suspension of face-to-face classes and the adoption of remote learning, preventive measures implemented in higher education institutions. This context made these students live a period of uncertainty regarding the future of their education28. Our results indicate a statistically significant association between loneliness and clinical signs and symptoms of stress, anxiety, and depression. A different study found sense of belonging, well-being, and acts of self-compassion to function as protective factors against these disorders20. A study conducted prior to social distancing found family stability  to cause positive effects on mental health22. Corroborating these findings, we verified that being single or widowed and unemployed incur a higher risk of severe/extreme psychological symptoms.Recent studies showed that married individuals have better health conditions than widowed, divorced, and single people30, we found reduction in family income during the pandemic to pose a risk factor for severe/extreme depression. Considering that all social classes may experience reduction in family income, this finding reflects economic instability rather than low-socioeconomic status31.Although studies conducted outside the social distancing context reported conflicting results regarding financial aspects29.As for employment status, individuals who were employed prior to social distancing and were furloughed  were considered to be at a risk of severe/extreme depression. Given that most of our study sample worked in the healthcare sector, we expected those who continued working to be in a risk group. A previous study found healthcare professionals to present a high risk for psychiatric disorders during this period due to increased feelings of vulnerability or loss, concerns with patients’ and family members’ health, virus spread, and changes in work and family routines18. This may also be explained by the fact that living at the epicenter of the disease could entail a risk factor for mental disorders. The current epicenter in Brazil is the city of São Paulo, located in the southeastern region. Living abroad was yet another relevant risk factor for stress and severe/extreme depression among Brazilians, increasing alongside the period of separation, possibly as a consequence of being distant from family.Individuals living in the Southeast region of Brazil were at greater risk of developing mental disorders than those living in the Northeastern. Corroborating our findings, a study conducted in California showed that continuous exposure to Covid-19-related information, especially in the major urban centers, increases stress and anxiety rates32. We found people who reported following no religion to be at a greater risk of presenting clinical signs and symptoms of severe/extreme depression. These results are supported by the literature, which indicates that religious involvement is correlated with better recovery and improve the ability to manage physical and mental illness34, especially during crises such as the Covid-19 pandemic. In this study, religion was self- stated, regardless of individuals’ degree of involvement.Religiosity plays a pivotal psychological role in believers’ lives by giving meaning to physical and psychological stress and providing comfort and support, particularly in difficult times35. These feelings trigger self-medication or excessive medication use, risk factors for the three mental disorders addressed in the study due to the increased vulnerability caused by the indiscriminate use of benzodiazepines36.We found individuals with previous history of anxiety and depression to present a greater risk of developing or worsening psychological symptoms during the pandemic. This finding is corroborated by another study, which suggests that this context is marked by boredom, disappointment, and irritability23. This might be explained by the fact that underlying diseases are strongly associated with severe forms of Covid-19 and increased hospital mortality.The presence of chronic diseases was significantly associated with clinical signs and symptoms of severe/extreme anxiety among our study sample, corroborating the results of a cross-sectional study conducted in China at the beginning of the year3. These findings are corroborated by a meta-analysis on the effects of exercise on mental disorders, which reported that physical activity could effectively control depression due to the excitatory effect on monoamine neurotransmitters and endorphins, and possibly improve self-esteem, self-perception, and personal satisfaction37.The practice of physical and leisure activities should be strongly encouraged during the pandemic context. We found participants who did not perform leisure or physical activities, as well as those who reduced these practices, to present a greater risk of mental disorders. The literature suggests that physical exercise and leisure activities appear to be interconnected, establishing an association between individuals’ cognitive and physical functioning and thus improving mental health1, added to the distress, fear, and implications of the disease, as well as frustration toward helplessness, boredom, and sadness1.As individuals in social isolation or quarantine are precisely those contaminated with or exposed to Covid-19, confinement necessarily results in separation, particularly from family members, loss of personal freedom, and changes in life habits29. In our study, individuals presenting symptoms, even without a confirmed diagnosis, were at greater risk of severe/extreme depression, suggesting that this new, unknown infection arise negative emotions, such as fear and anxiety.Studies found individuals who have or have had Covid-19 symptoms and those who had a family member or close relative diagnosed with the disease and shared the experience with them to be positively associated with anxiety, depression, and post-traumatic stress disorder (PTSD)13. However, our study has some limitations inherent to online data collection. The online approach of data collection was the most viable option in the current scenario, for preventing infection spread across investigators and the study population. A strength provided by such approach is that responses were obtained from all regions of the country. Although the final sample was heterogenous, respondents from the northeast region (investigators’ place of residence) were more prevalent. Even though 3,200 individuals comprised the study sample, such prevalence suggests the possibility of selection bias, given that the questionnaire was initially disclosed to the researchers’ contacts. However, the limitations imposed by the online data collection are minimized with the strict control over the eligibility criteria and with the sample size obtained.Our study questionnaire comprised two facets: the 21-item Depression, Anxiety and Stress Scale (DASS-21) validated for use in the Brazilian population; and other questionnaires collecting sociodemographic data, lifestyle habits, social distancing, and Covid-19. This second set of questionnaires was created by the authors based on a bibliographic research conducted in updated databases from regional, national, and international health agenciesYet another limitation to this study was including Brazilians living abroad. Although countries implemented different pandemic policies, all of them had restrictive social measures in place, and Brazilian individuals were facing further risk for being distant from their relatives.The literature suggests approaches different than that employed by us to questionnaires addressing signs and symptoms of stress, anxiety, and depression, making it difficult for us to compare the results with other studies. On the other hand, studies show these varying approaches have common ground, and the questionnaire was chosen based on researchers’ experience and the country validation.Considering that our main goal was to determine factors associated to mental disorders during social distancing due to the Covid-19 pandemic, the study sample also included individuals that, despite of the recommendations, might not have been socially distancing, or individuals who complied with the measure for varying length. This occurs either because they were considered essential workers or simply because they did not adhere to the government guidelines. Moreover, the observational study design increases difficulties in controlling for confounding factors. This issue was minimized with a multivariable analysis, which found no significant correlation between social distancing duration and the mental disorders analyzed.The DASS-21 questionnaire simply evaluates the prevalence of symptoms of stress, anxiety, and depression; it does not function as a diagnostic method. Considering that, a diagnosis on mental disorders requires a specialized medical evaluation.Our results indicate a high prevalence of clinical signs and symptoms of severe/extreme stress, anxiety, and depression during social distancing, increasing alongside the duration of the confinement period. We found various factors, many of which common to all three mental disorders, to be associated with the social distancing measures imposed during the Covid-19 pandemic.38, variables such as these can be used to develop federally-funded programs aiming at improving mental health by promoting psychotherapy activities and educational and recreational endeavors, stimulating physical exercise, and providing guidelines for the online workspace, particularly for helping women. Authorities should also promote and encourage video calls between Brazilians residing abroad and their friends and family as a means of communicating and establishing emotional bonds.Being of female gender, living abroad, practicing no leisure and physical activities, and being unemployed were the most relevant factors associated with signs and symptoms of stress, anxiety, and depression. As suggested by Brazilian researchersOur findings point to the need for implementing preventive measures aimed at reducing or stabilizing the prevalence of stress, anxiety, and depression. To avoid aggravating these mental disorders, early and effective action is crucial.


The lentil seed coat is a waste by-product still rich in phenolic compounds, specifically condensed tannins. The effect of different solvents, as well as different processes, namely conventional solid–liquid extraction (CSLE) and ultrasound-assisted extraction (UAE), on the extraction yield of specific phenolic compound classes was studied. Four empirical two-parameter models were examined to select the one that better fit the experimental data obtained under different operating conditions. Additionally, ultra-high-pressure liquid chromatography coupled to quadrupole-time-of-flight mass spectrometry (UHPLC-ESI/QTOF-MS) was employed to profile the phenolic compounds obtained under distinct extraction conditions. In the operative conditions adopted here, the bioactive compounds yield achieved using UAE was lower than that obtained with CSLE. The kinetics of polyphenols, flavonoids, and condensed tannins extraction from the lentil seed coat were successfully fitted to the power-law models, yielding mean values of the root mean square < 5.4%, standard error of estimation < 0.53, and coefficient of determination > 0.8. In addition, the UHPLC-ESI/QTOF-MS of the lentil seed coat extracts allowed the putative recognition of nearly 500 compounds, mainly flavonoids and phenolic acids. Lens culinaris L.) are an important source of protein and dietary fiber and are rich in micronutrients [Terminalia catappa L. kernel [Pulses are edible seeds and represent an important component of human nutrition in many regions worldwide . As withutrients ,3. Howevutrients . Howeverutrients ,6. Severutrients . As a coutrients . The feautrients . Typicalutrients . Howeverutrients . Therefoutrients ,12. To sutrients . Mathemautrients , the oil. kernel , and the. kernel , and som. kernel ,19,20,21Lentils of the Laird variety (Lens esculenta Moench) with a flattened lenticular shape and a green color were used. The lentil seed coat was separated from the cotyledon using a semi-industrial husker  at a rotor speed of 2300 rpm. The obtained hull was collected, vacuum-packed in plastic bags, and stored until extraction with conventional solid–liquid extraction or ultrasound-assisted extraction. All the solvents used were LC-MS grade, VWR (≥99.9%). 2O (70:30) (v/v), (b) EtOH:H2O (60:40) (v/v), and (c) deionized water. As the increased acidity supports the dissolution of phenolic compounds [v/v). After 15 h of extraction, the mixture was centrifuged at 3000× g for 10 min. After collecting the supernatant, the extracted lentil seed coat was re-extracted twice more, using each time 10 mL of the same solvent and following the same conditions. A suitable quantity of the fresh solvent was then added to the collected supernatants, bringing the final volume to 40 mL. Lastly, before the spectrophotometric assays, the extracts were filtered through a 0.45 µm PTFE membrane filter.Phenolic compounds were obtained from the lentil seed coat following the method reported by Zhang et al.  with sliompounds , all the2O (60:40) (v/v) and deionized water, both acidified with 0.1% HCl (v/v). After the extraction, the mixture was centrifuged at 3000× g for 10 min. A suitable quantity of the fresh solvent was then added to the collected supernatants, bringing the final volume to 40 mL. Lastly, before the spectrophotometric assays, the extracts were filtered through a 0.45 µm PTFE membrane filter.Ultrasound-assisted extraction with a 250 W rated output power and 40 kHz frequency was conducted on 200 mg of lentil seed coats in 40 mL of solvents at room temperature. The maximum extraction time was 2 h, based on the preliminary data obtained. Two different solvents were used: EtOH:H2CO3 was added. The reaction mixture was incubated at room temperature for 30 min. The absorbance was determined at 765 nm . The TPC was expressed as milligrams of gallic acid equivalents (GAE) per gram of dry weight (dw).The total phenolic compounds in the lentil seed coats extracted as above were evaluated, as described by Singleton and Rossi . In deta2 solution at room temperature. After 5 min, 60 μL of AlCl3 0.75 M was mixed and allowed to react for another 6 min before the addition of 400 μL of NaOH 0.5 M. The absorbance was determined at 500 nm using a spectrophotometer. The results were expressed as milligrams of catechin equivalents (CAE) per gram of dry weight (dw).The total flavonoid content (TFC) assay was performed, as reported by Dewanto et al. . In detaThe condensed tannin content (CTC) of the extracted samples was performed by the vanillin assay . Eight hTo evaluate the entire extraction process, four experimental kinetic models, generally implemented in modeling the extraction of solutes from solid materials, were employed to fit the experimental data, namely the parabolic diffusion, power-law, hyperbolic, and Elovich’s models. When applied to the extraction of plant materials, the parabolic diffusion equation can be written (Equation (1)) as: 0 is the washing coefficient instead, and A1 represents the diffusion rate constant (min−0.5).This model agrees with the two-step extraction process consisting of the washing of weakly bound material, which is instantaneously leached, followed by the diffusive release. In this sense, the parabolic diffusion model parameter A1 is the extraction rate at the beginning (min−1) and C2 is the constant related to the maximum extraction yield (min−1).The power-law model explains the extraction mechanism by the diffusion of solute through a nonswelling material (Equation (2)):0 and E1 are parameters of the Elovich equation. Elovich’s model (Equation (4)) refers to a logarithmic relation:The nonlinear kinetic equations and the linearized form of these models are presented in All particles were sphere-shaped with a uniform size.The solute component was uniformly distributed in the matrix.The diffusion coefficient of the solute components was constant.Solid particles were well distributed in the extracting solvent.The reported models were based on the following general assumptions:The TPC, TFC, and CTC yields of the lentil seed coat were calculated according to the following formula (Equation (5)):v/v), and a C18 column Agilent Zorbax eclipse plus . The acquisition of accurate masses was made at 30.000 FWHM in a positive full scan (100–1200 m/z range). The injection volume was 6 μL, and source conditions were as follows: nitrogen operated as a drying gas (8 L/min and 330 °C) and sheath gas (10 L/min and 350 °C), the capillary voltage was 3.5 kV, and the nozzle voltage was 300 V.The phytochemical profile of the different extracted matrix was investigated through an UHPLC-ESI/QTOF-MS, as previously reported by Rocchetti et al. . Brieflywww.phenol-explorer.eu, accessed 26 May 2021), and the compounds considered and included in the dataset were those identified within 100% of replications in at least one treatment.The Profinder B.06 software (from Agilent Technologies) was used for compound annotation from raw mass features, in accordance with the algorithm ‘find-by-formula.’ For annotation purposes, after post-acquisition filters, the entire isotope pattern  and a 5 ppm tolerance for mass accuracy were used to achieve a level 2 of confidence  of the Metabolomics Standard Initiative . The datAfterward, the compounds annotated were classified according to their respective phenolic class/subclass, and the cumulative abundances were determined from calibration curves of pure standard solutions . To this aim, cyanidin, catechin, quercetin, luteolin (flavonoids), ferulic acid (phenolic acids), tyrosol (low-molecular-weight compounds), sesamin (lignans), and resveratrol (stilbenes) were used for the quantification as the representative phenolic compounds, and the results were expressed as mg equivalents/kg dry weight (dw).p < 0.05. Statistical analyses were performed by XLSTAT . The Solver Microsoft Excel package  was used to evaluate data fittings using nonlinear regression. To assess the level at which models interpret the experimental data, the coefficient of determination (R2), root-mean-square (RMS), and standard error of estimation (SEE) were, respectively, calculated using the following equations: N is the number of experimental data points. cal and exp are the estimated and experimental values, respectively, in Equations (2) and (3), while x and y are the experimental and calculated values in Equation (4). dt is the change in time. The higher the R2 and the lower the RMS and SEE values, the better the goodness of fit.All data represent the means of at least three measurements. The means’ assessment was conducted using the ANOVA with a post hoc Tukey test at 2O) mixture under CSLE conditions are shown in Flavonoids and condensed tannins, the principal polyphenols in legume seeds, are broadly found in the lentil seed coat . The TPC2O) mixture or H2O as a solvent, both acidified with HCl 0.1 M, with CSLE and UAE was investigated. Although the methanolic mixture allowed a higher extraction of phenolic compounds from the lentil seed coat using CSLE, nowadays, more environmentally friendly processes with shorter extraction times and fewer organic solvents are of great concern. Therefore, the possibility to obtain a high yield using ethanol , the SEE and RMS. The greater the value of R2 and the lower the value of RMS and SEE, the better the goodness of fit. The statistical R2, RMS, and SEE results presented in 2 were higher than 0.622, the RMS values were lower than ±10% for all extraction conditions, and SEE was always lower than 1%. Thus, all the evaluated models may potentially be appropriate to understand the extraction of the phenolic compounds from the lentil seed coat using CSLE or UAE with different solvents for engineering purposes [2 increased in the following order: hyperbolic model → parabolic model → Elovich’s equation → power law model. 0 of the parabolic model, which symbolizes that the extraction yield was obtained immediately as the plant material was immersed into the solvent at t = 0. Higher values were obtained in CSLE for all the class compounds analyzed with organic solvents that allowed higher TPC and CTC yields. On the contrary, in UAE extraction, using water, a higher instantaneous TPC, TFC and CTC recovery was obtained. Although Elovich’s model showed a good fit, it did not show a good correlation for the CTC extraction (both in CSLE and UAE) and the CSLE with MeOH (R2 = 0.759), and 2 (0.809–1.000) and low RMS (%) (0.394–5.389) and SEE (0.002–0.528), which corroborated the model’s accuracy and suitability to describe the extraction. The power-law is the most relevant model for the extraction of a substance from a nonswelling device [These models have been previously implemented for the extraction of bioactive compounds from several matrices such as Asteraceae plant, pomegranate peels, barley, and chicory roots by-products ,42,43,44purposes . Howeverpurposes , it mustg device  with a dg device . Specifig device  investigg device  who workAn untargeted metabolomic methodology was used to comprehensively evaluate the phenolic composition of the singular lentil extracts, considering both the influence of the extraction solvents and the effect of ultrasound irradiation under different operating conditions.The whole list of compounds recorded in our experimental conditions is provided as Looking at the results, ethanol was the best solvent promoting the extraction of flavonoids (except for flavonols), phenolic acids, and stilbenes, while methanol-water extracts were particularly abundant in tyrosol equivalents. Indeed, adding water to organic solvents such as methanol and ethanol was found to better promote the extraction of phenolics; using water is not as effective as hydroalcoholic solutions, because polyphenols are broadly soluble in organic solvents with lower polarities than water .With regard to the ultrasound-assisted extraction (UAE), it is known that the efficiency may depend on several factors such as sonication amplitude, pulse cycle, extraction time, and the raw materials . Under oIn particular, the flavan-3-ols and flavonols were the classes showing the highest recovery after 120 min in both water 100% and aqueous ethanol (60:40) solutions, with increases of 1.2- and 1.3-folds, respectively. Moreover, the most effective extraction of low-molecular-weight compounds was achieved under aqueous conditions after 40 min of UAE (from 278.38 to 318.56 mg/kg). However, considering aqueous ethanol after 20 min of ultrasound application, the total phenolics had a 1.4-fold higher value than that of the control .Afterward, unsupervised hierarchical cluster (HCA) analysis was applied in order to naively compare the phenolic profiles under the different extraction solvents tested. As expected, two clusters were generated, with the organic solvents ethanol and methanol demonstrating a comparable phenolic profile, whilst water was separated in a second and more distinct cluster . InteresThen, the VIP approach (variable importance in projections) was applied to investigate those compounds most influenced by the extraction method used; only those compounds with a VIP score > 1.2 were considered and listed in the 2. Looking at the results, ethanol was the best solvent promoting the extraction of flavonoids, phenolic acids, and stilbenes, while methanol-water extracts were particularly abundant in tyrosol equivalents. Although the water was not as effective as hydroalcoholic solutions, it must be remembered that the choice of solvent should be based not only on the solubility and stability of the desired component in the system.Conventional solid-liquid extraction (CSLE) and ultrasound-assisted extraction (UAE) of the phenolic compounds  from the lentil seed coats were studied. Under the operative conditions adopted here, the bioactive compounds yield achieved using UAE were lower compared to the CSLE. The modeling studies on the kinetics of CSLE and UEA on lentil seed coat phenolic compounds displayed that the kinetics was highly conditioned from the process used, as well as on the extraction solvent. All the selected kinetic models fitted the experimental data quite well. However, the power-law model allowed us to obtain lower RMS and SEE and a higher RTaken together, the kinetic study represents an essential task in assessing the extraction process as it allows estimation of the cost-effectiveness. The obtained evidence is supportive when it is necessary to choose the suitable extraction method for natural matrices, including the scale-up to an industrial level. In this case, the scaling-up of the process will be reasonable. A convenient extraction procedure, based on the use of green solvents and cheap and abundant raw material that allows one to obtain an extract with a good concentration of bioactive compounds, was proposed.


Hemophagocytic lymphohistiocytosis syndrome (HLHS) is characterized by an immunological hyperactivation of cytotoxic T cells, natural killer cells, and macrophages, leading to the secretion of proinflammatory cytokines. HLHS associated with Visceral Leishmaniasis might be difficult to diagnose once symptoms are similar, resulting in the death of untreated patients. Our aim is to describe a series of cases of Visceral Leishmaniasis with HLHS admitted to a referral hospital for infectious diseases.All 115 cases of Visceral Leishmaniasis referred to a referral center for pediatric infectious diseases were reviewed to identify the cases of HLHS. Five cases (4.5%) were confirmed with HLHS and they presented fever, splenomegaly, cytopenia, hypertriglyceridemia or hypofibrinogenemia, increased ferritin and hemophagocytosis in the bone marrow.It important to rule out HLHS in children with infectious diseases that do not respond adequately to treatment or in patients with severe symptoms, especially in leishmaniasis endemic areas.% HLHS associated with VL might be difficult to diagnose once symptoms are similar, resulting in death if left untreated.,Visceral leishmaniasis (VL) is a systemic disease that affects the reticuloendothelial system, caused by 3, neutrophils <1,000/mm3); 4) hypertriglyceridemia (≥265mg/dL) and/or hypofibrinogenemia (≤1.5g/L); 5) hemophagocytosis in the bone marrow, spleen, or lymph nodes; 6) low or absent NK cell activity; 7) hyperferritinemia (ferritin ≥500μ/L); and 8) elevation of CD25 ≥2,400U/mL.The diagnostic criteria for HLHS are based on clinical, laboratory, and morphological data and must include 5 of the following 8 criteria:–The treatment of primary HLHS consists of immunosuppressive drugs. However, in cases associated to VL, patients should be treated with liposomal amphotericin (LA) and, only if they do not respond well to specific treatment, should immune modulation be considered.It should be emphasized that in an endemic area for VL such as Belo Horizonte,Fundação Hospitalar do Estado de Minas Gerais This was a descriptive study performed in Belo Horizonte, Minas Gerais, Brazil, from January 2016 to April 2018. All cases of VL referred to pediatric infectious diseases center were reviewed and cases of HLHS were identified according to diagnostic criteria.A total of 115 cases of VL were assisted at the referral center and five cases of HLHS were described. Clinical manifestations are presented below and clinical and laboratory criteria for HLHS are described in Leishmania spp. and hemophagocytosis. She presented liver failure, and lipossomal amphotericin B and corticosteroid were started. She was treated with cefepime and vancomycin due to sepsis . After 12 days, she was discharged from the hospital and continued oral corticosteroid therapy.A 14-month-old female infant was admitted with fever, hepatosplenomegaly, bicytopenia, hypoxemia, and increased serum ferritin. A myelogram revealed Leishmania spp., and amastigotes were detected in a myelogram, which also presented hemophagocytosis. Complete treatment with lipossomal amphotericin was started and cefepime was used due to fever-associated neutropenia. In addition, he underwent transfusion of platelets, red blood cells, and albumin. After a diagnosis of HLHS, corticotherapy was started. The patient recovered after 3 days of treatment.A 7-month-old male infant presented with fever for 10 days associated with hepatosplenomegaly, bicytopenia, pallor, anasarca, and increased ferritin. Immunochromatographic rapid test with rK39 antigen was positive for Leishmania and the myelogram presented Leishmania spp with hemophagocytosis. Then, glucantime was started. As fever persisted, on day 8, nonresponse to glucantime was considered and it was replaced by lipossomal amphotericin. Due to the fever and neutropenia, cefepime was also started. After further testing, the patient fulfilled 5 of the 8 HLHS criteria. After clinical and laboratory improvement, cefepime was suspended on day 9 and the patient was discharged on day 12.A one-year-old male child was admitted with a history of continuous fever for 3 months and hepatosplenomegaly, anemia, hypertriglyceridemia, increased ferritin, and hemodynamic instability, requiring blood transfusion. Immunochromatographic rapid test with rK39 antigen was positive for Leishmania spp. and hemophagocytosis. On day 2, lipossomal amphotericin was started and a blood transfusion was necessary. On day 3, HLHS was confirmed. However, on day 5, she presented clinical worsening, with respiratory insufficiency, hypoalbuminemia, and a corticosteroid was initiated. On day 8, the patient improved, with resolution of fever and neutropenia and a negative blood culture resulted in discontinuation of the antimicrobial. She was discharged on day 15 and continued oral corticosteroid therapy for 8 weeks.A female infant aged 2 years was admitted presenting with fever for 4 days, oliguria, generalized edema, and hematochezia for 2 days. She was pale, with hepatosplenomegaly and anasarca. Laboratory tests revealed pancytopenia. Cefepime was started due to neutropenia and clinical instability, A myelogram revealed A 9-year-old male child was admitted to the hospital with fever and headache for 12 days associated with polyarthralgia. He had autoimmune hepatitis treated with prednisone, azathioprine, and vitamin D. He was pale with hepatosplenomegaly and jaundice. On day 2, the patient was diagnosed with VL by means of an immunochromatographic rapid test with rK39 antigen and hemophagocytic syndrome and lipossomal amphotericin was started. The child also presented increased ferritin, hypertriglyceridemia, and hypofibrinogenemia. Because of the febrile neutropenia in an immunosuppressed patient, cefepime was started on day 5. On day 8, lipossomal amphotericin was completed. Due to clinical and laboratory improvement, the antibiotic was finished and the child was discharged on day 11.In this 2-year survey at a referral center for infectious diseases, 4.4% of patients with lipossomal amphotericin were suspected to have HLHS. Of these, only one did not meet the criteria needed for the diagnosis owing to a borderline triglyceride level . In a reHLHS is a syndrome of immune hyperactivation and release of cytokines, characterized by clinical signs and symptoms of severe and uncontrolled inflammation. Although rare in adults, it is frequently seen in the pediatric population caused by several infectious diseases.A 5-year review of the literature revealed only 32 case reports of HLHS, with most cases being secondary to Epstein-Barr virus infection.VL should be ruled out in all children with HLHS, as the symptoms of the infection may overlap with those of the HLH syndrome and could result in death if left untreated.Leishmania spp. 3 months later and lipossomal amphotericin was started. Domínguez-Pinilla et al.Although the treatment of HLHS includes immunosuppressive drugs, it can be resolved with specific treatment of leishmaniasis.In the present series, all patients received lipossomal amphotericin, and only 1 patient did not receive corticosteroid therapy. However, 2 patients had severe disease with hemodynamic instability and another patient used corticosteroids due to autoimmune hepatitis. Immunosuppressive drugs should be reserved for patients who do not respond within the first 2 to 3 days of specific treatment.,Both HLHS and VL are often associated with neutropenia, inducing infections and antibiotics are used during febrile neutropenia,This report reinforces the importance of suspecting HLHS in children with infectious diseases that do not respond adequately to treatment or with severe symptoms, especially in leishmaniasis endemic areas.


The majority (95%) of participants were overweight with a high body fat and low muscle and bone mass, leading to a high prevalence of OSA (>50%), OA (13%), and AD (26%). There were only a few participants with SA, and they were not analyzed. The highest extracellular water/total body water ratio was observed in the OSA participants, indicating a heightened inflammatory state. Participants in all three body composition categories had a similar nutrient intake, with protein, fiber, omega-3 fatty acids, and almost all micronutrients being far below recommendations. In conclusion, a high prevalence of OSA among NHRs accompanied by a poor dietary intake, could place these residents at a very high risk for COVID-19 infections. Therefore, optimization of body composition and nutritional status should be included along with standard medical care in order to provide better health maintenance, particularly in the COVID-19 era.The population of older adults, especially those living in the nursing homes, is growing. The sedentary lifestyle and possible poor nutrition in nursing homes place residents (NHRs) at risk for body composition impairments, malnutrition, and, subsequently, numerous chronic diseases. The aim of this study was to assess body composition (including body fluids) and dietary intake in NHRs. The association between osteosarcopenic adiposity syndrome (OSA) and its components, osteopenic adiposity (OA), sarcopenic adiposity (SA), and adiposity-only (AD), and specific macro- and micro-nutrients was evaluated as well. The study included 84 participants (82.1% women), aged 65.3–95.2 years. Body composition was assessed with an advanced bioelectrical impedance device BIA-ACC Older adults are the fastest growing segment of the population throughout the world . In the Several years ago, the simultaneous occurrence of low bone mass (osteopenia/osteoporosis), low muscle mass/strength (sarcopenia/dynapenia), and increased/redistributed adiposity was termed osteosarcopenic adiposity syndrome (OSA) ,8. OSA pApart from bone, muscle, and adipose tissue deteriorations , body fluids also change with aging, particularly extracellular water (ECW) . Recent 6, B9, B12, A, D, E and K; and an excess of sodium and phosphorus [These age-related changes in body composition influence nutrient metabolism and requirements in older adults . A low iosphorus ,12,30,31osphorus ,33.Based on the demographic context characterized by a growing aging population and especially by those living in nursing homes, the need for studies in this area has become extremely relevant. Presently, the need is even more intensified by the high risk for older adults, especially those living in nursing homes, to succumb to COVID-19 infections ,33,34,35n = 355), living in the nursing home for ≥6 months were invited to participate in the study. Ethical approval was granted by the Ethics Committee of the Institute of Medical Research and Occupational Health (100–21/18–10), and the study was designed in accordance with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. The nurses from the nursing home informed the residents about the study aims and methods, and listed those individuals who were interested and were >65 years. Residents who could sit to stand independently (with or without aid) and provide informed consent were invited to participate in the study. Those who had a contradiction to bioimpedance measurement  and who had cancer related cachexia were not invited. The study commenced in February 2020 but had to be abruptly stopped in March due to COVID-19 pandemic lockdowns.All residents over 65 years of age from a nursing home in Zagreb, Croatia , prescription drugs, and smoking, as well as their dietary intake. Regarding fractures, an osteoporotic fracture was defined as either a fracture resulting from a low-impact fall in participants with a diagnosis of osteoporosis or from a fall resulting in a low-trauma fracture . The num®, Marcon-Venice, Italy) measuring fat-free mass ; total bone mass (kg); volumetric bone mineral density ; skeletal muscle mass ; fat mass ; intramuscular adipose tissue ; and ECW and ICW, as percentage of TBW, the latter being the % of body weight. The normal range of the ECW/TBW is provided by the manufacturer, and in a healthy state it should fall between 0.360 to 0.390 [The participants’ body weight and height were measured with a digital scale and stadiometer  to the nearest 0.1 kg and 0.1 cm, respectively, without shoes and in indoor clothing. Body composition was assessed with a portable, advanced bioelectrical impedance device BIA-ACC , and the manual “Quantitative models of food and meals”  with phot-test was used, and for non-parametric data, the Mann–Whitney U test was used to compare the differences between groups (men vs. women). The Chi-square test was used to compare the frequency of participants with osteoporotic fractures, limited mobility, smoking, and medications usage (men vs. women). ANOVA with a post hoc Scheffe test was used to analyze the differences in the anthropometric, body composition, and dietary parameters among participants stratified into groups according to body composition categories. In addition, a Pearson correlation for normally distributed continuous variables and Spearman correlation for not-normally distributed continuous variables, as well as the regression analyses, controlled for various confounders, were conducted for closer data examination. Analyses were done with IBM SPSS Statistics v. 23.0, released 2015  and Statistica 13.0 software . The level of significance was set at p < 0.05.The results are shown as mean ± standard deviation (unless noted otherwise) for continuous variables and as percentages for categorical variables. The distribution of variables was tested using the Shapiro Wilk test. Bivariate analyses were performed depending on the type and distribution of variables. For normally distributed data (continuous variables), the independent Student’s n = 84 participants were recruited, of whom 82.2% were women (80.7 ± 6.8 years) and 17.8% men (86.3 ± 3.5 years). The average time spent in the nursing home was 4.5 and 3.7 years for women and men, respectively . The intake of all other vitamins and minerals was below the EFSA recommendation for both women and men (n = 34) were taking vitamin and/or mineral supplements , of which the most commonly taken were vitamins B1 (17.7%), B2 (20.6%), B3 (23.5%), B6 (52.9%), and C (50.0%), as well as minerals calcium (17.7%), magnesium (76.5%), and potassium (12.8%). Two men were taking calcium supplements only.Regarding dietary intake, women and men had a similar energy, macronutrient, and micronutrient intake . Based o and men . This lon = 4, all women) of participants without adiposity and 2.3%  with sarcopenic adiposity. They were excluded from the further analyses because of the small numbers (The classification of participants into body composition categories is presented in  numbers .p < 0.001) and lower ICW  compared with the participants in AD (ECW 49.8 ± 2.2% of TBW and ICW 50.2 ± 2.2% of TBW) and OA (ECW 50.6 ± 1.6% of TBW and ICW 49.4 ± 1.6% of TBW), the latter two having a similar distribution . In the logistic regression model (controlled for age), with OSA as a dependent variable and ECW, protein intake, years in residency, and medication index as predictors, significance was reached for ECW and years in residency. The odds ratio for ECW was 1.5 , while the odds ratio for years in residency was 1.1 .In the correlational analyses, only a few nutrients showed statistical significance with body composition components (not presented). For example, in both the AD and OA groups, protein intake was positively related to ICW and bone mass and negatively to ECW, while fat intake was negatively related to bone mass. Additionally, in the OA group, fiber intake was positively related to muscle and bone mass, and vitamins A and K were positively to bone mass. In the OSA group, protein intake was positively related to the T-score, and carbohydrate intake to BMI, body fat, and intramuscular fat  and its association with dietary intake in older adults living in nursing homes, as well as to examine the possible influence of ECW (indicator of inflammation) in relation to body composition. Our results showed a high prevalence of OSA (>53%) in both women and men , with ECAs aging uniquely influences many physiological functions, the most observable are those regarding body composition changes, including loss of bone, loss of muscle mass and strength, and increased and/or redistributed adiposity, all potentially leading to the most advanced deterioration, known as OSA syndrome ,8. PreseReports from some studies indicate that a higher ECW/TBW ratio can be predictive of sarcopenia ,15. DuriThe issue of body fluids also attracts attention in the field of obesity and nutrition, as a high ECW/ICW is noted in obese individuals due to obesity-related edema and the hormonal responses of adipose tissue itself . It has The analysis of dietary intake revealed no significant difference in energy and macronutrient intake between participants with OSA, OA, and AD, except for protein , which was highest in the AD group, despite that it was below the recommendations for all groups . A similProtein intake, expressed as g/day, was significantly lower in both women and men with OSA compared with those in other body composition categories in the studies by Kim et al. ,53, but 1, B2, and B3; calcium; phosphorus; sodium; and iron was similar in participants from all groups [A few studies have reported on the intake of micronutrients in participants with OSA and compared it with participants with milder body composition abnormalities  ,51,53. Al groups . The intl groups . In addiThe reason for the reported discrepancy in the literature for energy and nutrient intake could be explained by the use of different methodologies to assess dietary intake and the inherent limitations in any of the dietary surveys, well recognized and discussed previously , which dBesides the poor body composition and high prevalence of OSA, our study revealed reasons for concern regarding inadequate nutrition among NHRs, especially considering that long-term inadequate nutrient intake may promulgate the occurrence of OSA ,31, as wAn interesting study was performed in one Belgian nursing home, where the energy and protein content of the served food was compared with the actual food consumed by NHRs, reporting that residents consumed just fragments of the meals, and the actual energy and protein  content of the consumed food was considerably less than what was served . DespiteOverall, it seems that an insufficient energy and protein intake is common among residents of nursing homes ,60,63, wDietary fat intake should amount to 20–35% of energy intake, and saturated fatty acid should be as low as possible  or below1, B2, B3, B6 and C above recommendations. Men were below recommendations for all vitamins, except for vitamins B1 and B3. Of note is the extremely low intake of vitamin D in all participants  were below recommendations, phosphorus was above recommendations, while sodium was in accordance with recommendations  for bothndations , Figure ndations ,69, partndations . These nVitamin A intake in our participants was also below recommendations. It appears that both a too high and too low intake of vitamin A could impair bone health in older individuals ,71; therVitamin D has received a lot of attention recently, not just for its undisputed role in bone metabolism and muscle functioning, but also as a potential anti-inflammatory and immunity-enhancing nutrient . Higher Calcium and magnesium are well known for their role in bone, muscle, and adipose tissue health ,80, and As around 90% of zinc in the human body is located in muscle, bone, skin, and hair, it is obvious that a zinc deficiency may result in impaired bone formation, as well as in muscle and connective tissue structure and metabolism disturbances, as reviewed earlier . In a KoConsidering the relatively long-time residency in nursing homes (4.5 and 3.7 years on average for women and men respectively), and the multiple comorbidities and medications in NHRs , a healtThe limitations of our study are its cross-sectional nature, its relatively small sample size, and the unequal number of women and men, although this is not uncommon in other studies. The small sample size might have particularly been a case in subgroup analyses in which the number of participants was even more reduced and thus a statistical power could not be reached for possible significance. On the other hand, in a smaller study, it is possible to recruit a more uniform sample with strict exclusion/inclusion criteria and to measure an array of important biological and lifestyle variables, and to account for them as potential confounders. Even more so, it is possible to collect subject-based recalls and self-reported data more accurately, as participants could be individually instructed and interact with researchers, as was the case in our study.Another limitation is the dietary assessment via 24-h dietary recall, which, as with any other dietary record, has inherent shortcomings . HoweverWe did not collect the data on the physical activity of NHRs. However, as a recent study in almost 300 NHRs in Zagreb reported , all nurOur study began in February 2020 just before the COVID-19 pandemic caused massive lockdowns and the closure of nursing homes to visitors. Therefore, we present these results as a pilot study with the aim to continue and build upon it as soon as the restriction measures are lifted. In view of the COVID-19 pandemic and the recognized higher risk of infection among NHRs ,33, we bOur results indicate the high prevalence of OSA among NHR and significantly elevated ECW in OSA participants compared with those with other body composition impairments, indicating a higher inflammatory state for the former. The analysis of dietary intake showed no significant difference in energy, macronutrient, and micronutrient intake between participants with OSA, OA, and AD, except for protein intake  and selenium. However, all participants had a poor dietary intake, with most of the nutrients below the recommended levels, including protein, omega-3 fatty acids, and fiber, as well as most of micronutrients, particularly vitamin D. Providing adequate and nutritious food to older adults in nursing homes is crucial to ensure nutritional adequacy, optimal body composition, and to maintain their health and a good quality of life. In order to avoid a deficiency of macro- and micro-nutrients in NHRs, possible dietary supplements should be considered.