Correlation of serum YKL-40, 25(OH)D3 and HMGB1 expression levels with infection types and disease assessment in neonatal pneumonia

Objective: To investigate the serum levels of human cartilage glycoprotein 39 (YKL-40), 25-hydroxy vitamin D3 [25-hydroxy vitamin D3, 25 (OH)D3] and high mobility group protein B1 (high mobility group protein B1, HMGB1) level changes in the diagnosis of neonatal pneumonia infection type and the application of disease assessment. Methods: A total of 105 children with NP who were admitted to the Department of Neonatology, Longhua District People's Hospital of Shenzhen from January to December 2020 were selected as the research objects. According to different infectious pathogens, they were divided into a bacterial pneumonia group of 40 cases and a non-bacterial pneumonia group of 65 cases. According to the severity of the disease, they could be divided into 69 cases of mild pneumonia group and 36 cases of severe pneumonia group, and 85 healthy newborns were selected as the control group during the same period. Serum levels of YKL-40, 25 (OH)D3 and HMGB1 were detected by enzyme-linked immunosorbent assay. ROC curve was used to analyze the differential diagnosis value of YKL-40, 25 (OH) D3 and HMGB1 for NP alone or in combination. Spearman rank correlation was used the relationship between serum YKL-40, 25 (OH) D3 and HMGB1 levels and the severity of the disease in children with NP was analyzed. Results: The serum levels of YKL-40 (46.39 +or- 8.36 ng/ml, 40.28 +or- 8.47 ng/ml)and HMGB1 (23.38 +or- 5.66 ng/ml, 17.32 +or- 4.18 ng/ml) in the bacterial pneumonia groups and non-bacterial pneumonia groups were significantly higher than those in the control group (30.49 +or- 6.35 ng/ml, 12.56 +or- 3.22 ng/ml), and the differences were statistically significant (F=939.480, 99.507, all P < 0.05), while bacterial and non-bacterial pneumonia groups serum 25 (OH) D3 (12.76 +or- 3.57 g/L, 18.33 +or- 4.21 g/L) levels were significantly lower than those in the control group (19.76 +or- 4.87 g/L), and the difference was statistically significant (F=225.752, P < 0.05). The serum levels of YKL-40 (52.56 +or- 9.68 ng/ml) and HMGB1 (26.74 +or- 4.57 ng/ml) in the severe group were significantly higher than those in the mild group (16.63 +or- 5.32 ng/ml, 9.63 +or- 2.38 ng/ml) and the control group (11.63 +or- 3.32 ng/ml, 6.34 +or- 2.06 ng/ml), the differences were all statistical significance (F=265.331, 55.426, all P < 0.05), and serum 25 (OH) D3 (9.76 +or- 3.54 g/L, 31.16 +or- 5.01 g/L)levels in the severe and mild were significantly lower than control groups (35.16 +or- 5.88 g/L) (F=55.426, P < 0.05) . The results of Spearman rank correlation analysis showed that the disease severity was positively correlated with serum YKL-40 and HMGB1 levels (r=0.727, 0.210, all P < 0.05), but negatively correlated with 25 (OH) D3 levels (r= -0.566, P < 0.05). The results of ROC curve analysis showed that the combined detection of YKL-40, 25 (OH) D3 and HMGB1 had the highest efficacy in diagnosing NP, the AUC was 0.912 (95%CI: 0.864-0.932), the sensitivity and specificity were 96.34%, 85.72%, respectively. In the single detection of each index, the differences in AUC were statistically significant (Z=0.746, 2.843, 3.662, all P < 0.05). The combined detection of the three had the highest diagnostic efficiency in distinguishing neonatal bacterial pneumonia from non-bacterial pneumonia, and its AUC was 0.894 (95%CI: 0.832-0.941), the sensitivity and specificity were 97.26%, 80.66%, respectively. Which was higher than the single test of each index, and the difference in AUC was statistically significant (Z=1.573, 3.228, 2.689, all P < 0.05). Conclusion: Serum levels of YKL-40, 25 (OH) D3 and HMGB1 had important clinical value in diagnosis of NP infection types and in reflecting the severity of children's disease, the combined detection of the three has better clinical diagnostic performance.

Effective multidimensional approach for practical management of the emergency department in a COVID-19 designated children's hospital in east China during the Omicron pandemic: a cross-sectional study.

Background: Characterized by rapid transmission but lower severity, the new Omicron wave brought about an acute increase in local corona virus disease 2019 (COVID-19) cases in Shanghai, followed by stricter infection prevention and control strategies. Inevitably, more time was required for emergency consultation and treatment of children with critical illnesses. Therefore, a multidimensional approach was designed to streamline the emergency service and reduce the incidence of nosocomial infection of sever acute respiratory syndrome coronavirus 2 (SARS-CoV-2) during the Omicron wave for the emergency department (ED) at the Children's Hospital of Fudan University (CHFU). Methods: A multidimensional approach was implemented in the ED to help achieve a balance between the demand for emergency services and pandemic control, consisting of ED layout adjustment; electronic screening (E-screening) measures; standard management processes for patients, medical staff, and goods transfer; reliable disinfection measures; and a surveillance system for infection prevention and control. To evaluate the effect of the management strategy, the data on nosocomial infection cases and occupational exposure episodes among staff in the ED were collected. The demographic and clinical characteristics of level I/II children by the five-level pediatric triage tool and their mean duration of stay in the resuscitation room were collected. Results: There were 12,114 ED visitors from March 1 to May 31 in 2022, among which 53.24% were medical emergencies (6,449/12,114) and 46.76% were surgical emergencies (5,665/12,114). Twenty-nine patients were sent to the buffer zone, four of whom were transferred to the pediatric intensive care unit (PICU) because of the critical situation. Six patients tested positive for COVID-19 after entering ED, including three in the buffer zone and three in the ED clinic, causing a temporary closure of the ED for disinfection. There were no reports on medical care delays, unintended deaths, staff with COVID-19 infection, or occupational exposures to COVID-19. Conclusions: Our findings highlight the effectiveness of the multidimensional approach, which can simultaneously meet the emergency care needs of patients as well as pandemic prevention and control. However, the results were obtained against the proportional decrease in clinic visitors due to the Shanghai lockdown. Dynamic assessment and further optimization may be adopted to cope with the pre-pandemic visit volume.

Experience of the homecare of children on automatic peritoneal dialysis during the COVID-19 outbreak: A qualitative descriptive study.

AIMS: To describe the home care experience, challenges and coping strategies of caregivers with children on automatic peritoneal dialysis (PD) in mainland China during the early stage of the COVID-19 outbreak. DESIGN: A qualitative descriptive approach was adopted. Semi-structured telephone interviews were conducted among 14 families with children on automatic peritoneal dialysis from February 2nd to 10th, 2020. The care routine, stress and coping strategies of caregivers of children on peritoneal dialysis were collected. The data were analysed using thematic analysis. METHODS: Four key themes were defined: (1) concerns about PD treatment intertwined with worries about COVID-19; (2) retaining a sense of normality in the middle of the challenges; (3) staying safe; and (4) staying positive and carrying on. RESULTS: Families with children on automatic PD addressed the stress from COVID-19 and its containment measures by closely adhering to COVID-19 preventative measures, actively adjusting mentality and maintaining a sense of normality during the outbreak. This implies that healthcare staff need to be more aware of the complex medical needs of families with children on automatic PD, advocate for them and facilitate their navigation through the repurposed healthcare system.

Genetic engineering of baculovirus-insect cell system to improve protein production.

The Baculovirus Expression Vector System (BEVS), a mature foreign protein expression platform, has been available for decades, and has been effectively used in vaccine production, gene therapy, and a host of other applications. To date, eleven BEVS-derived products have been approved for use, including four human vaccines [Cervarix against cervical cancer caused by human papillomavirus (HPV), Flublok and Flublok Quadrivalent against seasonal influenza, Nuvaxovid/Covovax against COVID-19], two human therapeutics [Provenge against prostate cancer and Glybera against hereditary lipoprotein lipase deficiency (LPLD)] and five veterinary vaccines (Porcilis Pesti, BAYOVAC CSF E2, Circumvent PCV, Ingelvac CircoFLEX and Porcilis PCV). The BEVS has many advantages, including high safety, ease of operation and adaptable for serum-free culture. It also produces properly folded proteins with correct post-translational modifications, and can accommodate multi-gene- or large gene insertions. However, there remain some challenges with this system, including unstable expression and reduced levels of protein glycosylation. As the demand for biotechnology increases, there has been a concomitant effort into optimizing yield, stability and protein glycosylation through genetic engineering and the manipulation of baculovirus vector and host cells. In this review, we summarize the strategies and technological advances of BEVS in recent years and explore how this will be used to inform the further development and application of this system.

Genetic engineering of baculovirus-insect cell system to improve protein production

The Baculovirus Expression Vector System (BEVS), a mature foreign protein expression platform, has been available for decades, and has been effectively used in vaccine production, gene therapy, and a host of other applications. To date, eleven BEVS-derived products have been approved for use, including four human vaccines [Cervarix against cervical cancer caused by human papillomavirus (HPV), Flublok and Flublok Quadrivalent against seasonal influenza, Nuvaxovid/Covovax against COVID-19], two human therapeutics [Provenge against prostate cancer and Glybera against hereditary lipoprotein lipase deficiency (LPLD)] and five veterinary vaccines (Porcilis Pesti, BAYOVAC CSF E2, Circumvent PCV, Ingelvac CircoFLEX and Porcilis PCV). The BEVS has many advantages, including high safety, ease of operation and adaptable for serum-free culture. It also produces properly folded proteins with correct post-translational modifications, and can accommodate multi-gene– or large gene insertions. However, there remain some challenges with this system, including unstable expression and reduced levels of protein glycosylation. As the demand for biotechnology increases, there has been a concomitant effort into optimizing yield, stability and protein glycosylation through genetic engineering and the manipulation of baculovirus vector and host cells. In this review, we summarize the strategies and technological advances of BEVS in recent years and explore how this will be used to inform the further development and application of this system.

Characterization and immunogenicity of SARS-CoV-2 spike proteins with varied glycosylation.

The ongoing coronavirus disease-19 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has drastically changed our way of life and continues to have an unmitigated socioeconomic impact across the globe. Research into potential vaccine design and production is focused on the spike (S) protein of the virus, which is critical for virus entry into host cells. Yet, whether the degree of glycosylation in the S protein is associated with vaccine efficacy remains unclear. Here, we first optimized the expression of the S protein in mammalian cells. While we found no significant discrepancy in purity, homogeneity, or receptor binding ability among S proteins derived from 293F cells (referred to as 293F S-2P), 293S GnTI- cells (defective in N-acetylglucosaminyl transferase I enzyme; 293S S-2P), or TN-5B1-4 insect cells (Bac S-2P), there was significant variation in the glycosylation patterns and thermal stability of the proteins. Compared with the partially glycosylated 293S S-2P or Bac S-2P, the fully glycosylated 293F S-2P exhibited higher binding reactivity to convalescent sera. In addition, 293F S-2P induced higher IgG and neutralizing antibody titres than 293S or Bac S-2P in mice. Furthermore, a prime-boost-boost regimen, using a combined immunization of S-2P proteins with various degrees of glycosylation, elicited a more robust neutralizing antibody response than a single S-2P alone. Collectively, this study provides insight into ways to design a more effective SARS-CoV-2 immunogen.

A Bacterially Expressed SARS-CoV-2 Receptor Binding Domain Fused With Cross-Reacting Material 197 A-Domain Elicits High Level of Neutralizing Antibodies in Mice.

The Coronavirus disease 2019 (COVID-19) pandemic presents an unprecedented public health crisis worldwide. Although several vaccines are available, the global supply of vaccines, particularly within developing countries, is inadequate, and this necessitates a need for the development of less expensive, accessible vaccine options. To this end, here, we used the Escherichia coli expression system to produce a recombinant fusion protein comprising the receptor binding domain (RBD) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2; residues 319-541) and the fragment A domain of Cross-Reacting Material 197 (CRM197); hereafter, CRMA-RBD. We show that this CRMA-RBD fusion protein has excellent physicochemical properties and strong reactivity with COVID-19 convalescent sera and representative neutralizing antibodies (nAbs). Furthermore, compared with the use of a traditional aluminum adjuvant, we find that combining the CRMA-RBD protein with a nitrogen bisphosphonate-modified zinc-aluminum hybrid adjuvant (FH-002C-Ac) leads to stronger humoral immune responses in mice, with 4-log neutralizing antibody titers. Overall, our study highlights the value of this E. coli-expressed fusion protein as an alternative vaccine candidate strategy against COVID-19.

Landscapes and dynamic diversifications of B-cell receptor repertoires in COVID-19 patients.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the pandemic of coronavirus disease 2019 (COVID-19). Great international efforts have been put into the development of prophylactic vaccines and neutralizing antibodies. However, the knowledge about the B cell immune response induced by the SARS-CoV-2 virus is still limited. Here, we report a comprehensive characterization of the dynamics of immunoglobin heavy chain (IGH) repertoire in COVID-19 patients. By using next-generation sequencing technology, we examined the temporal changes in the landscape of the patient's immunological status and found dramatic changes in the IGH within the patient's immune system after the onset of COVID-19 symptoms. Although different patients have distinct immune responses to SARS-CoV-2 infection, by employing clonotype overlap, lineage expansion, and clonotype network analyses, we observed a higher clonotype overlap and substantial lineage expansion of B cell clones 2-3 weeks after the onset of illness, which is of great importance to B-cell immune responses. Meanwhile, for preferences of V gene usage during SARS-CoV-2 infection, IGHV3-74 and IGHV4-34, and IGHV4-39 in COVID-19 patients were more abundant than those of healthy controls. Overall, we present an immunological resource for SARS-CoV-2 that could promote both therapeutic development as well as mechanistic research.

A Novel Modified-Curcumin Promotes Resolvin-Like Activity and Reduces Bone Loss in Diabetes-Induced Experimental Periodontitis.

PURPOSE: Clinically, it is challenging to manage diabetic patients with periodontitis. Biochemically, both involve a wide range of inflammatory/collagenolytic conditions which exacerbate each other in a "bi-directional manner." However, standard treatments for this type of periodontitis rely on reducing the bacterial burden and less on controlling hyper-inflammation/excessive-collagenolysis. Thus, there is a crucial need for new therapeutic strategies to modulate this excessive host response and to promote enhanced resolution of inflammation. The aim of the current study is to evaluate the impact of a novel chemically-modified curcumin 2.24 (CMC2.24) on host inflammatory response in diabetic rats. METHODS: Type I diabetes was induced by streptozotocin injection; periodontal breakdown then results as a complication of uncontrolled hyperglycemia. Non-diabetic rats served as controls. CMC2.24, or the vehicle-alone, was administered by oral gavage daily for 3 weeks to the diabetics. Micro-CT was used to analyze morphometric changes and quantify bone loss. MMPs were analyzed by gelatin zymography. Cell function was examined by cell migration assay, and cytokines and resolvins were measured by ELISA. RESULTS: In this severe inflammatory disease model, administration of the pleiotropic CMC2.24 was found to normalize the excessive accumulation and impaired chemotactic activity of macrophages in peritoneal exudates, significantly decrease MMP-9 and pro-inflammatory cytokines to near normal levels, and markedly increase resolvin D1 (RvD1) levels in the thioglycolate-elicited peritoneal exudates (tPE). Similar effects on MMPs and RvD1 were observed in the non-elicited resident peritoneal washes (rPW). Regarding clinical relevance, CMC2.24 significantly inhibited the loss of alveolar bone height, volume and mineral density (ie, diabetes-induced periodontitis and osteoporosis). CONCLUSION: In conclusion, treating hyperglycemic diabetic rats with CMC2.24 (a tri-ketonic phenylaminocarbonyl curcumin) promotes the resolution of local and systemic inflammation, reduces bone loss, in addition to suppressing collagenolytic MMPs and pro-inflammatory cytokines, suggesting a novel therapeutic strategy for treating periodontitis complicated by other chronic diseases.

Cross-neutralizing antibodies bind a SARS-CoV-2 cryptic site and resist circulating variants.

The emergence of numerous variants of SARS-CoV-2, the causative agent of COVID-19, has presented new challenges to the global efforts to control the COVID-19 pandemic. Here, we obtain two cross-neutralizing antibodies (7D6 and 6D6) that target Sarbecoviruses' receptor-binding domain (RBD) with sub-picomolar affinities and potently neutralize authentic SARS-CoV-2. Crystal structures show that both antibodies bind a cryptic site different from that recognized by existing antibodies and highly conserved across Sarbecovirus isolates. Binding of these two antibodies to the RBD clashes with the adjacent N-terminal domain and disrupts the viral spike. Both antibodies confer good resistance to mutations in the currently circulating SARS-CoV-2 variants. Thus, our results have direct relevance to public health as options for passive antibody therapeutics and even active prophylactics. They can also inform the design of pan-sarbecovirus vaccines.

Point-of-care training program on COVID-19 infection prevention and control for pediatric healthcare workers: a multicenter, cross-sectional questionnaire survey in Shanghai, China.

BACKGROUND: To evaluate the effectiveness of training on knowledge and practice of infection prevention and control (IPC) among pediatric health care workers (HCW) in Shanghai, China, in the context of COVID-19 pandemic. METHODS: An online training program was designed by the Shanghai Pediatric Clinical Quality Control Center (SPQCC) during the early phase of COVID-19 pandemic on disease knowledge and practice of IPC. Training took place in the 81 partner hospitals affiliated with SPQCC. A multicenter, cross-sectional questionnaire survey was designed with a 25-item self-administered questionnaire to evaluate the knowledge gained from the training. Stratified-random sampling was used to select HCW according to three professionals (i.e., pediatricians, nurses and administrators) within each partner hospital. Awareness and knowledge of COVID-19 and its related infection control and practice were assessed by comparing survey results between different types of hospitals, professionals and professional ranks. A higher survey score meant that the respondent was more prepared and knowledgeable about COVID-19 and its infection control measures. RESULTS: Completed questionnaires were returned from 1,062 subjects (385 pediatricians, 410 nurses, and 267 administrators), giving a response rate of 96.5%. Overall, awareness of clinical information related to COVID-19, importance of personal hygiene and isolation policy was high among the respondents. No statistical difference of scores on knowledge of COVID-19, IPC and relevant practice between the tertiary and peripheral hospitals. Among all respondents, middle-ranked health care personnel were most knowledgeable and achieved the highest score. CONCLUSIONS: Majority of pediatric HCW showed good recognition and practice in infection protection and control measures. The online training was able to achieve its aim to enhance knowledge and awareness and could have contributed to the zero infection rate among HCW caring for confirmed COVID-19 cases in Shanghai.

Current Progress and Challenges in the Design and Development of a Successful COVID-19 Vaccine

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is still a worldwide concern, with little to no sign of a decreasing trend. There is a general consensus that normal life will be hampered until a safe and effective vaccine strategy is available and globally administered. Numerous countries have accelerated the clinical trials process for the development of a successful COVID-19 treatment, with over 200 candidates presently available for testing against SARS-CoV-2. Here, we provide an overview of the COVID-19 vaccine candidates currently in development, discuss the scientific and practical challenges associated with COVID-19 vaccine design, and share the potential strategies that could be exploited for vaccine design success.

SARS-CoV-2 spike produced in insect cells elicits high neutralization titres in non-human primates.

The current coronavirus disease 2019 (COVID-19) pandemic was the result of the rapid transmission of a highly pathogenic coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), for which there is no efficacious vaccine or therapeutic. Toward the development of a vaccine, here we expressed and evaluated as potential candidates four versions of the spike (S) protein using an insect cell expression system: receptor binding domain (RBD), S1 subunit, the wild-type S ectodomain (S-WT), and the prefusion trimer-stabilized form (S-2P). We showed that RBD appears as a monomer in solution, whereas S1, S-WT, and S-2P associate as homotrimers with substantial glycosylation. Cryo-electron microscopy analyses suggested that S-2P assumes an identical trimer conformation as the similarly engineered S protein expressed in 293 mammalian cells but with reduced glycosylation. Overall, the four proteins confer excellent antigenicity with convalescent COVID-19 patient sera in enzyme-linked immunosorbent assay (ELISA), yet show distinct reactivities in immunoblotting. RBD, S-WT and S-2P, but not S1, induce high neutralization titres (>3-log) in mice after a three-round immunization regimen. The high immunogenicity of S-2P could be maintained at the lowest dose (1 µg) with the inclusion of an aluminium adjuvant. Higher doses (20 µg) of S-2P can elicit high neutralization titres in non-human primates that exceed 40-times the mean titres measured in convalescent COVID-19 subjects. Our results suggest that the prefusion trimer-stabilized SARS-CoV-2 S-protein from insect cells may offer a potential candidate strategy for the development of a recombinant COVID-19 vaccine.

The evaluation of online course of Traditional Chinese Medicine for MBBS international students during the COVID-19 epidemic period.

BACKGROUND: During the COVID-19 epidemic period, Traditional Chinese Medicine (TCM) course for international students of Medical Bachelor, Bachelor of Surgery (MBBS) program in Zhejiang University has shifted from traditional classroom to online environment. This study aimed to investigate MBBS international students' perception on online TCM course, and to assess the online learning efficacy. METHODS: A total of 84 MBBS international students attending course of "Basic Traditional Chinese Medicine" during 2020 academic years at Zhejiang University were enrolled in this study. A quantitative questionnaire was respectively completed before and after the TCM course using a pretest-post-test design. By means of two online learning platforms, Learning in ZJU and DingTalk, TCM course was broadcast in both live and archived format to students. RESULTS: A total of 48 participants completed both baseline and follow-up questionnaires. The majority of participants preferred face-to-face classroom learning (26, 54.17% of total) when compared with online learning. Students felt that the course had brought in much benefits (mean 3.88, SD 0.87), and they were satisfied with the course content (mean 3.83, SD 0.95). Students' TCM related knowledge and their behaviors of discussion and consulting were significantly improved by online TCM course (all P < 0.001). Students' awareness of the necessity of TCM education and their feeling of difficulty in learning TCM were significantly strengthened (P = 0.042, 0.025, respectively). CONCLUSION: Online learning is a good alternative for TCM course of MBBS international students when classroom learning is suspended, whereas it cannot replace the need for onsite and face-to-face learning.

A quickly, effectively screening process of novel corona virus disease 2019 (COVID-19) in children in Shanghai, China.

BACKGROUND: A recent cluster of pneumonia cases in China was caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We report the screening and diagnosis of corona virus disease 2019 (COVID-19) in our hospital. METHODS: Developed a procedure for the identification of children cases with COVID-19 in outpatient and emergency department of our hospital, then we observed how this process works. RESULTS: (I) There were 56 cases considered suspected cases, and 10 cases were confirmed as COVID-19. (II) Of the 10 confirmed COVID-19 cases admitted in our hospital, 5 were males and 5 were females, aged from 7 months to 11 years, the average age is 6.0±4.2 years, 6 cases were mild pneumonia, the others were upper respiratory tract infection. (III) We followed up 68 patients in isolation at home until symptoms disappeared. Non were missed in the patient's first visit. The sensitivity of this method is 100% and the specificity is 71.3%. CONCLUSIONS: Our screening process works well, and it is also necessary to establish a screening network in the hospital.