[Preparation and characterization of chicken anti-human B7-H4 IgY polyclonal antibody].

Objective To prepare anti-human B7 homolog 4 (B7-H4) egg yolk immunoglobulins (IgY) polyclonal antibody and establish a double-antibody sandwich ELISA for the detection of soluble B7-H4 (sB7-H4) protein in human serum. Methods Bioinformatics was used to screen specific B cell epitope peptides of human sB7-H4. New Hyland Grey laying hens were immunized with these peptides, and the eggs from the immunized hens were collected to purify chicken anti-human B7-H4 IgY antibody. The purity, concentration and titer of the antibody were detected, and its specificity and function of the antibodies were verified by using ELISA, Western blot analysis and flow cytometry, respectively. A double-antibody sandwich ELISA was established to detect sB7-H4 in clinical samples by using the IgY antibody. Comparative detection was performed using a commercialized ELISA kit on the same set of clinical samples. Results The chicken anti-human B7-H4 IgY antibodies were successfully prepared and proven to be highly specific for the human B7-H4 protein. The ELISA established with the IgY polyclonal antibody detected significantly higher levels of soluble B7-H4 in the serum of patients with ovarian cancer and benign ovarian tumors compared to healthy controls. These results were consistent with the detection results obtained using a commercialized ELISA kit. However, the ELISA with IgY antibody exhibited higher sensitivity than the commercialized kit. Conclusion The chicken polyclonal antibody against human B7-H4 IgY is successfully prepared, and a double-antibody sandwich ELISA suitable for detecting sB7-H4 protein in human serum is established.

Development of a Novel Recombinant Full-Length IgY Monoclonal Antibody against Human Thymidine Kinase 1 for Automatic Chemiluminescence Analysis on a Sandwich Biotin-Streptavidin Platform for Early Tumour Discovery.

Serum thymidine kinase 1 protein (STK1p) concentration has been used successfully as a reliable proliferating serum biomarker in early tumour discovery and clinical settings. It is detected by an enhanced chemiluminescence (ECL) dot blot assay with the biotin-streptavidin (BSA) platform (a gold standard) based on chicken anti-human thymidine kinase 1 IgY polyclonal antibody (hTK1-IgY-pAb). However, ECL dot blotting is a semiautomatic method that has been limited to large-scale applications due to the differences among batches of antibodies from individual hens, and the skill level of operation technicians sometimes results in unstable STK1p values. Therefore, a highly stable recombinant chicken full-length IgY monoclonal antibody in combination with a fully automated sandwich biotin-streptavidin (sandwich-BSA) platform was developed. Hens were immunized with 31-peptide, a key sequence of human TK1 (hTK1), before constructing an immune phage display scFv library. Finally, a recombinant full-length IgY monoclonal antibody (hTK1-IgY-rmAb#5) with high-affinity binding with human recombinant TK1 (rhTK1) (3.95 × 10-10 mol/L), high sensitivity with hTK1 calibrators (slope of linear curve: 89.98), and high specificity with low/elevated STK1p (r ≈ 0.92-0.963) was identified. hTK1-IgY-rmAb#5 showed a specific immune response with thymidine kinase 1 (TK1) in TK1-positive/negative cell lysates by Western blotting and immunohistochemistry (IHC) in normal and cancer tissues. In particular, the detection of TK1 serum samples from health centres showed a high coincidence rate (r = 0.988, n = 90) between hTK1-IgY-rmAb#5 and hTK1-IgY-pAb and between the semiautomatic ECL dot blot BSA platform and the novel automatic chemiluminescence sandwich-BSA platform (r = 0.857, n = 292). hTK1-IgY-rmAb#5 is stable and highly sensitive for detecting the lowest STK1p value at 0.01 pmol/L (pM). The accuracy is high (SD < 2.5%) between different batches. It is easy to use the novel hTK1-IgY-rmAb#5 on a new automatic chemiluminescence sandwich-BSA platform. It will be beneficial for large-scale health screenings.

Is serum thymidine kinase 1 a prognostic biomarker in primary tumor location of colorectal carcinomas?

AIM: To assess whether serum thymidine kinase 1 (STK1p), CEA and CA19.9 can be used as prognostic biomarkers in the primary tumor location (PTL) of colorectal carcinoma (CRC). Additional clinical factors of TNM stage, pathological grade, age and sex were also included. METHODS: STK1p was determined by an ECL-dot-blot assay, and CEA/CA19.9 was determined by an automatic electrochemiluminescence analyzer in a retrospective presurgery of right-colon carcinoma (R-CC, n = 90), left-colon carcinoma (L-CC, n = 128) and rectal carcinoma (RC, n = 270). Prognostic factors were evaluated by COX and overall survival (OS). RESULTS: The multivariate-COX and OS in relation to the prognostic factors of PTL in CRC were different and complex. An elevated STK1p value was significantly associated with poor OS in RC (P = 0.002) and L-CC (P = 0.037) but not in R-CC (P > 0.05). Elevated CEA (P≈.000) and CA19.9 (P≈.000) were significantly associated with poor OS in RC but not in L-CC and R-CC. Multivariate-COX showed that STK1p (P = 0.02, HR = 1.779, 95%CI 1.30-7.582), CEA (P = 0.001, HR = 2.052, 95%CI 1.320-3.189), CA19.9 (P≈.000, HR = 2.574, 95%CI 1.592-4.162) and TNM-stage (P≈.000, HR = 2.368, 95%CI 1.518-3.694) were independent prognostic factors in RC, while TNM-stage was an independent prognostic factor only in R-CC (P = 0.011, HR = 3.139, 95% CI 1.30-7.582) and L-CC (P≈.000, HR = 4.168, 95%CI 1.980-8.852). Moreover, elevated STK1p was significantly more sensitive (P < .001) for predicting mortality than CEA and CA19.9. No correlation was found between STK1p, CEA or AFP. CONCLUSION: Combining TNM stage and suitable biomarkers, STK1p provides further reliable information on the survival of PTL of CRC.

Novel neutralizing chicken IgY antibodies targeting 17 potent conserved peptides identified by SARS-CoV-2 proteome microarray, and future prospects.

Introduction: An approach toward novel neutralizing IgY polyclonal antibodies (N-IgY-pAb) against SARS-CoV-2 S-ECD was developed. Material and methods: The novel N-IgY-pAb and its intranasal spray response against the wild type ("'WH-Human 1") SARS-CoV-2 virus, variants of Delta or Omicron were up to 98%. Unique virus peptides binding to N-IgY-pAb were screened by a SARS-CoV-2 proteome microarray. Results: Seventeen mutation-free peptides with a Z-score > 3.0 were identified as potent targets from a total of 966 peptides. The new findings show that one is in the RBM domain (461LKPFERDISTEIYQA475 ), two are in the NTD domain (21RTQLPPAYTNSFTRG35, 291CALDPLSETKCTLKS305) four are in the C1/2-terminal (561PFQQFGRDIADTTDA575,571DTTDAVRDPQTLEIL585,581TLEILDITPCSFGGV595, 661ECDIPIGAGICASYQ675 ), three are in the S1/S2 border (741YICGDSTECSNLLLQ755, 811KPSKRSFIEDLLFNK825, 821LLFNKVTLADAGFIK835) one target is in HR2 (1161SPDVDLGDISGINAS1175) and one is in HR2-TM (1201QELGKYEQYIKWPWY1215). Moreover, five potential peptides were in the NSP domain: nsp3-55 (1361SNEKQEILGTVSWNL1375), nsp14-50 (614HHANEYRLYLDAYNM642, ORF10-3 (21MNSRNYIAQVDVVNFNLT38, ORF7a-1(1MKIILFLALITLATC15) and ORF7a-12 (1116TLCFTLKRKTE121). Discussion and conclusion: We concluded that the N-IgY-pAb could effectively neutralize the SARS-CoV-2. The new findings of seventeen potent conserved peptides are extremely important for developing new vaccines and "cocktails" of neutralizing Abs for efficient treatments for patients infected with SARS-CoV-2.

Serum thymidine kinase 1 protein concentration for predicting early progression and monitoring the response to TACE in hepatocellular carcinomas: a network meta-analysis.

AIM: A meta-analysis was conducted to evaluate the clinical significance of serum thymidine kinase 1 protein concentration (STK1p) in distinguishing between hepatocellular carcinomas (HCC) and non-HCC for predicting early progression and monitoring the response to transarterial chemoembolization in HCC. MATERIALS & METHODS: A total of 24 eligible studies were included, containing 1849 HCC patients and 1069 healthy subjects. RESULTS: The STK1p level significantly increased from normal controls to benign/pre-HCC and HCC (p < 0.0001). STK1p also increased significantly in sub-malignant groups: control being the lowest, followed consecutively by hepatic hemangioma, hepatitis B virus infection and hepatic cirrhosis (p < 0.05). After 1 month of transarterial chemoembolization treatment, STK1p level declined significantly, by 44.4% (p < 0.0001). CONCLUSION: STK1p is a useful prognostic biomarker in HCC.

A meta-analysis of serological thymidine kinase 1 as a marker for colorectal benign and malignant tumor risk assessment.

The present study investigated whether a concentration of serum thymidine kinase 1 (STK1p) could be used to distinguish between healthy individuals, patients with colorectal benign tumors and individuals with colorectal cancer (CRC). The effectiveness of surgery on patients with CRC was monitored. A total of 20 publications containing patients with CRC (n=1,836), patients with colorectal benign tumors (n=774) and healthy controls (n=1,701) were analysed in the present meta-analysis. The publications were collected from PubMed, Embase, CENTRAL, CNKI, Wanfang, VIP and SinoMed databases from January 1, 2009 until August 31, 2019. Articles were analyzed according to sensitivity (Forest plot) and publication bias (Begg's plot, Egger's linear regression) using fixed or random effect models to calculate the weighted mean difference. Study quality was checked using the Newcastle-Ottawa Scale Document Quality Assessment Scale. The meta-analysis followed the PRISMA statement. The results revealed that STK1p significantly distinguished healthy individuals and those with colorectal benign tumors from patients with CRC, and from patients with benign tumors (P<0.000001). STK1p levels also decreased by 40% following surgery (P<0.0001), which corresponded to half-life of ~1 month. The quality of the present study was high and no bias was identified among publication. It was concluded that STK1p was a reliable biomarker for the early detection of benign lesions, which may therefore prevent their future development into colorectal malignancies. STK1p may also be used for the clinical dynamic monitoring of the effectiveness of surgery in patients with CRC. Combining STK1p with colorectal-associated biomarkers, in addition to the determination of tumor stage and grade may therefore be of use.

STK1p as a prognostic biomarker for overall survival in non-small-cell lung carcinoma, based on real-world data.

AIM: A prospective investigation of serum thymidine kinase 1 concentration (STK1p) was performed to evaluate its prognostic value in patients with non-small-cell lung carcinoma (NSCLCs). PATIENTS & METHODS: The STK1p values of 127 patients were determined by an enhanced chemiluminescent dot blot assay. The patients were recruited from March 2011 to December 2017. RESULTS: Kaplan-Meier plot showed that patients with elevated STK1p values had worse overall survival (OS), especially patients of early/middle stages. Multi-variable COX regression showed that STK1p value and combined treatment surgery + chemotherapy were independent prognostic factors for favorable OS. CONCLUSION: STK1p is helpful in predicting OS of early/middle stages (I-IIIA) NSCLCs patients following a nonrandomized individual adapted treatment, but is may be not recommended in advanced stages (IIIB + IV) of NSCLCs.

Association Between Neighborhood Cohesion and Cancer Screening Utilization in Chinese American Older Adults.

This study aims to examine the association between neighborhood cohesion and cancer screening utilization in a community-dwelling Chinese American older population. Data were drawn from the Population Study of Chinese Elderly including 3159 Chinese American older adults aged 60 and above in the greater Chicago area. Cancer screening utilization was assessed by asking whether participants had undergone colon, breast, cervical, or prostate cancer screening. Neighborhood cohesion was measured through six questions. Logistic regression analysis showed that greater neighborhood cohesion was associated with higher likelihood of utilizing a mammogram (OR 1.32, 95% CI 1.14-1.52), a Pap test (OR 1.22, 95% CI 1.06-1.41), but not of a blood stool test (OR 1.10, 95% CI 0.98-1.23), a colonoscopy (OR 1.05, 95% CI 0.94-1.17), and a PSA test (OR 1.13, 95% CI 0.95-1.34). This study suggests positive associations between neighborhood cohesion and breast and cervical cancer screening utilization among a Chinese American older population.

Association Between Social Engagement and Cancer Screening Utilization in a Community-Dwelling Chinese American Older Population.

Objectives: This study aims to examine the association between social engagement and cancer screening utilization among a Chinese American older population. Method: Data were drawn from the Population Study of Chinese Elderly (PINE). In total, 3,157 Chinese older adults enrolled in this study. Cancer screening utilization was assessed by asking whether participants had utilized cancer screenings. Social engagement was measured with 16 questions. Results: After controlling for potential confounders, a higher level of social engagement was associated with increased utilization in blood stool test (OR 1.03, [1.02, 1.05]), colonoscopy (OR 1.02, [1.01, 1.03]), mammography (OR 1.06, [1.05, 1.08]), Pap test (OR 1.04, [1.03, 1.06]), and prostate specific antigen (PSA) test (OR 1.03, [1.01, 1.05]). Compared with those with low levels of social engagement, older adults with high levels of social engagement were more likely to have utilized a blood stool test (OR 1.85, [1.46, 2.35]), a colonoscopy (OR 1.37, [1.09, 1.72]), a mammography (OR 3.05, [2.25, 4.14]), and a Pap test (OR 1.99, [1.49, 2.66]), but not more likely to have utilized a PSA test (OR 1.40, [0.97, 2.03]). Conclusion: This study underscores the association between active social engagement and increased utilization of cancer screening among a Chinese American older population. Improving social engagement could be helpful in promoting cancer screening utilization.

Neighborhood Disorder Is Associated With Greater Risk for Self-Neglect Among Chinese American Older Adults: Findings From PINE Study.

Objective: This study aims to examine the association between neighborhood disorder with self-neglect among a community-dwelling Chinese American older population. Method: Data were extracted from the Population Study of Chinese Elderly (PINE), a cross-sectional study of 3,157 participants living in the greater Chicago area. Self-neglect was assessed with systematic observations of participants' personal and home environment. Neighborhood disorder was measured through eight questions. Logistic regression was used to examine the association between neighborhood disorder and self-neglect. Results: After adjusting for potential confounding factors, every one point higher in neighborhood disorder score was significantly associated with greater risk for overall self-neglect (odds ratio [OR] = 1.13, [1.11, 1.16]), mild self-neglect (OR = 1.14, [1.11, 1.17]), and moderate/severe self-neglect (OR = 1.12, [1.09, 1.16]). Regarding the phenotypes of self-neglect, every one point higher in neighborhood disorder score was significantly associated with greater risk for hoarding (OR = 1.17, [1.14, 1.20]), personal hygiene (OR = 1.15, [1.12, 1.19]), house in need of repair (OR = 1.12, [1.10, 1.15]), unsanitary conditions (OR = 1.12, [1.09, 1.15]), and inadequate utilities (OR = 1.10, [1.05, 1.15]). Discussion: This study underscores the significant association between disordered neighborhood and greater risk of self-neglect among Chinese American older adults. Improving neighborhood disorder problems could benefit for elder self-neglect prevention.

Increased Oxidative Damage of RNA in Early-Stage Nephropathy in db/db Mice.

To evaluate RNA oxidation in the early stage of diabetic nephropathy, we applied an accurate method based on isotope dilution high-performance liquid chromatography-triple quadruple mass spectrometry to analyze the oxidatively generated guanine nucleosides in renal tissue and urine from db/db mice of different ages. We further investigated the relationship between these oxidative stress markers, microalbumin excretion, and histological changes. We found that the levels of 8-oxo-7,8-dihydroguanosine (8-oxoGuo) and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) were increased in the urine and renal tissue of db/db mice and db/db mice with early symptoms of diabetic nephropathy suffered from more extensive oxidative damage than lean littermate control db/m mice. Importantly, in contrast to the findings in db/m mice, the 8-oxoGuo levels in the urine and renal tissue of db/db mice were higher than those of 8-oxodGuo at four weeks. These results indicate that RNA oxidation is more apparent than DNA oxidation in the early stage of diabetic nephropathy. RNA oxidation may provide new insight into the pathogenesis of diabetic nephropathy, and urinary 8-oxoGuo may represent a novel, noninvasive, and easily detected biomarker of diabetic kidney diseases if further study could clarify its source and confirm these results in a large population study.

Association Between Neighborhood Cohesion and Self-Neglect in Chinese-American Older Adults.

OBJECTIVES: To examine the association between neighborhood cohesion and risk of self-neglect in a community-dwelling Chinese-American older population. SETTING: Community. PARTICIPANTS: Chinese-American older adults aged 60 and older interviewed from 2011 to 2013 (N = 3,159). DESIGN: Data were drawn from the Population Study of Chinese Elderly, a cross-sectional community-engaged study in the greater Chicago area. MEASUREMENTS: Self-neglect was assessed with systematic observations of a participant's personal and home environment. Neighborhood cohesion was measured using six questions. RESULTS: After controlling for potential confounders, greater neighborhood cohesion was significantly associated with lower risk of overall self-neglect (odds ratio (OR) = 0.87, 95% confidence interval (CI) = 0.77-0.98) and moderate to severe self-neglect (OR = 0.70, 95% CI = 0.58-0.85) but not significantly associated with mild self-neglect (OR = 0.94, 95% CI = 0.82-1.09). Regarding the phenotypes of self-neglect, greater neighborhood cohesion was significantly associated with lower risk of poor personal hygiene (OR = 0.80, 95% CI = 0.67-0.96) and need for home repair (OR = 0.70, 95% CI = 0.60-0.83) but not significantly for hoarding (OR = 1.04, 95% CI = 0.89-1.21), unsanitary conditions (OR = 0.88, 95% CI = 0.76-1.02), and inadequate utilities (OR = 1.00, 95% CI = 0.77-1.31). CONCLUSION: This study highlights the association between greater neighborhood cohesion and lower risk of overall self-neglect in Chinese-American older adults. Enhancing neighborhood cohesion may enhance elder self-neglect prevention and intervention.

High-Resolution Analyses of Human Leukocyte Antigens Allele and Haplotype Frequencies Based on 169,995 Volunteers from the China Bone Marrow Donor Registry Program.

Allogeneic hematopoietic stem cell transplantation is a widely used and effective therapy for hematopoietic malignant diseases and numerous other disorders. High-resolution human leukocyte antigen (HLA) haplotype frequency distributions not only facilitate individual donor searches but also determine the probability with which a particular patient can find HLA-matched donors in a registry. The frequencies of the HLA-A, -B, -C, -DRB1, and -DQB1 alleles and haplotypes were estimated among 169,995 Chinese volunteers using the sequencing-based typing (SBT) method. Totals of 191 HLA-A, 244 HLA-B, 146 HLA-C, 143 HLA-DRB1 and 47 HLA-DQB1 alleles were observed, which accounted for 6.98%, 7.06%, 6.46%, 9.11% and 7.91%, respectively, of the alleles in each locus in the world (IMGT 3.16 Release, Apr. 2014). Among the 100 most common haplotypes from the 169,995 individuals, nine distinct haplotypes displayed significant regionally specific distributions. Among these, three were predominant in the South China region (i.e., the 20th, 31st, and 81sthaplotypes), another three were predominant in the Southwest China region (i.e., the 68th, 79th, and 95th haplotypes), one was predominant in the South and Southwest China regions (the 18th haplotype), one was relatively common in the Northeast and North China regions (the 94th haplotype), and one was common in the Northeast, North and Northwest China (the 40th haplotype). In conclusion, this is the first to analyze high-resolution HLA diversities across the entire country of China, based on a detailed and complete data set that covered 31 provinces, autonomous regions, and municipalities. Specifically, we also evaluated the HLA matching probabilities within and between geographic regions and analyzed the regional differences in the HLA diversities in China. We believe that the data presented in this study might be useful for unrelated HLA-matched donor searches, donor registry planning, population genetic studies, and anthropogenesis studies.

Oxidative damage to RNA and expression patterns of MTH1 in the hippocampi of senescence-accelerated SAMP8 mice and Alzheimer's disease patients.

Mammalian MTH1 protein, a MutT-related protein, catalyzes the hydrolysis of 8-oxo-7,8-dihydroguanosine triphosphate (8-oxoGTP) to monophosphate, thereby preventing incorporation of 8-oxo-7,8-dihydroguanine (8-oxoguanine) into RNA. In this study, we applied immunohistochemistry to follow the expression of MTH1 and the amount of 8-oxoguanine in RNA during aging. There were increased amounts of 8-oxoguanine in RNA in the CAl and CA3 subregions of hippocampi of 8- and 12-month-old SAMP8 mice, which exhibited early aging syndromes and declining learning and memory abilities compared to those of age-matched control SAMR1 mice. The expression levels of MTH1 in the hippocampi of 8- and 12-month-old SAMP8 mice were significantly lower than those of control mice. Therefore, in this mouse model, age-related accumulation of 8-oxoguanine in RNA is correlated with decreased expression of MTH1. Increased amounts of 8-oxoguanine in the RNA, and decreased expression of MTH1 were also observed in the hippocampi of patients suffering from Alzheimer's disease. These results suggest that MTH1 deficiency might be a causative factor for aging and age-related disorders.

Age-related alterations in the expression of MTH2 in the hippocampus of the SAMP8 mouse with learning and memory deterioration.

MutT-related proteins degrade 8-oxo-7,8-dihydrodeoxyguanosine triphosphate (8-oxo-dGTP), a mutagenic substrate for DNA synthesis in the nucleotide pool, thereby preventing DNA replication errors. MTH2 (Mut T homolog 2), which belongs to this family of proteins, possesses 8-oxo-7,8-dihydro-2'-deoxyguanosine triphosphatase (8-oxo-dGTPase) activity and appears to function in the protection of the genetic material from the untoward effects of endogenous oxygen radicals. To examine the roles of MTH2 in the aging process, we used the senescence-accelerated prone mouse 8 (SAMP8), which exhibits early aging syndromes and declining abilities of learning and memory. Immunohistochemical and western blot analysis revealed that the level of MTH2 protein in the hippocampus of the SAMP8 mouse progressively decreases beginning from four months after birth, whereas no such change was observed in the control senescence-accelerated resistant mouse 1 (SAMR1). Under these conditions, 8-oxoguanine accumulates in the nuclear DNA in the CA1 and CA3 subregions of the hippocampus of SAMP8 in an age-dependent manner. In SAMR1 mice, accumulation of 8-oxoguanine in the DNA was not observed. These results suggest that the MTH2 deficiency might be one of the causative factors for accelerated aging.

Development of a method for concentrating and purifying SARS coronavirus RNA by a magnetic bead capture system.

Severe acute respiratory syndrome (SARS) is a recently emerged infectious disease caused by a novel coronavirus, which has been designated the SARS coronavirus (SARS-CoV). To date, molecular assays for the detection of SARS-CoV has focused mainly on reverse transcriptase-PCR (RT-PCR) analysis of specimens. However, RT-PCR assays currently available have low sensitivity during the early stage of the disease in which the viral load in specimens is very low. A method for concentrating and purifying SARS-CoV RNA by a magnetic bead capture system was developed and followed by an RT-PCR assay in this study with the goal of improving the sensitivity of the RT-PCR method. This approach takes advantage of the cooperative interaction between adjacently hybridized oligonucleotides. A capture probe was covalently coupled to magnetic beads and a second probe, which anneals adjacent to the capture probe site, was prehybridized in solution to the target. It was shown that, when applied to SARS RNA samples, the sensitivity of nucleic acid capture RTPCR was about 10-fold greater than routine RT-PCR. This nucleic acid capture system was effective in improving the sensitivity of the RT-PCR, due to enriching and purifying SARS-CoV RNA. The method will be helpful for the early detection of the SARS-associated coronavirus.