Predictive Validity of the Johns Hopkins Fall Risk Assessment Tool for Older Patients in Stroke Rehabilitation.

The aim of this retrospective, cross-sectional, observational study was to assess the frequency of falls and evaluate the predictive validity of the Johns Hopkins Fall Risk Assessment Tool (JHFRAT) among patients aged ≥65 years, transferred to the rehabilitation ward of a university hospital. The predictive ability was assessed using receiver operating characteristic curve analysis, and the optimal threshold was established using the Youden index. We analyzed the overall cohort (N = 175) with subacute stroke and the subgroup with a low unaffected handgrip strength (HGS; men: <28 kg, women: <18 kg). Overall, 135/175 patients (77.1%) had a low HGS. The fall rate was 6.9% overall and 5.9% for patients with a low HGS. The JHFRAT predictive value was higher for patients with a low HGS than that for the overall cohort, but acceptable in both. The optimal cutoff score for the overall cohort was 11 (sensitivity, 67%; specificity, 68%), whereas that for the subgroup was 12 (sensitivity, 75%; specificity: 72%). These results are expected to aid nurses working in rehabilitation wards in more effectively utilizing JHFRAT outcomes for post-stroke older patients with a low HGS and contribute to the development of more appropriate fall prevention strategies for high-risk patients in the future.

Efficacy and safety of oral sulfate tablet vs. polyethylene glycol and ascorbate for bowel preparation in children.

Background and aim: Bowel preparation for pediatric colonoscopy presents several challenges. However, no bowel preparation regimen is universally preferred for children. We aimed to investigate the efficacy and safety of oral sulfate tablet (OST) in pediatric bowel preparation. Methods: This study retrospectively analyzed data from children who received 2l of polyethylene glycol and ascorbate (PEG/Asc) or OST for bowel preparation between 2021 and 2023. A comparative analysis was conducted between the two groups. Results: A total of 146 patients were included (2l PEG/Asc: 115, 73.0% vs. OST: 31). No significant difference was observed in the total BBPS score (median 8.0 vs. 8.0, P = 0.152) and the total OBPS score (median 5.0 vs. 3.0, P = 0.152) between the two groups. No significant difference was noted in the ratio of a bubble score of 0 (73.0% vs. 93.5%, P = 0.132). The incidence of abdominal pain was significantly lower in the OST group (32.2% vs. 3.2%, P = 0.002). The VAS score for overall satisfaction was significantly higher in the OST group (4.0 vs. 7.0, P < 0.001). For the next colonoscopy bowel preparation, a higher proportion of patients in the OST group showed a willingness to use the same preparation regimen (33.9% vs. 83.9%, P < 0.001). Conclusion: OST was as efficacious and safe as 2 L of PEG/Asc for pediatric bowel preparation. The satisfaction level was higher with OST than with 2 L of PEG/Asc. OST may be considered a good alternative for children with poor compliance during bowel preparation.

Regional Disparities in the Infant Mortality Rate in Korea Between 2001 and 2021.

BACKGROUND: The infant mortality rate (IMR) has been considered an important indicator of the overall public health level. Despite improvements in recent decades, regional inequalities in the IMR have been reported worldwide. However, there are no Korean epidemiological studies on regional disparities in the IMR. METHODS: We extracted causes of death data from the Statistics Korea through the Korean Statistical Information Service database between 2001 and 2021. The total and regional IMRs were calculated to determine regional disparities. Based on causes of death and using Seoul as a reference, the excess infant deaths and population attributable fractions (PAFs) were calculated for 15 other metropolitan cities and provinces. The average annual percent changes by region from 2001 to 2021 were obtained using a joinpoint regression program. To assess inequities in IMR trends, the rate ratios (RRs) and rate differences (RDs) of the 15 regions were calculated by dividing the study period into period 1 (2001-2007), period 2 (2008-2014), and period 3 (2015-2021). RESULTS: The overall IMR in Korea was 3.64 per 1,000 live births, and the IMRs in the 14 regions were relatively higher than that in Seoul, with RRs ranging from 1.15 (95% confidence interval [CI], 1.04, 1.27) in Jeju-do to 1.62 (95% CI, 1.54, 1.71) in Daegu, over the total study period. Significant differences in infant deaths by region were observed for all causes of death, with PAFs ranging from 2.2% (95% CI, 1.7, 2.6) in Gyeonggi-do to 38.4% (95% CI, 38.1, 38.6) in Daegu. The leading cause of excess infant deaths was perinatal problems. The IMR disparities in the relative and absolute measures decreased from 1.44 (1.34, 1.54) to 1.21 (1.10, 1.31) for RRs and from 0.79 (0.63, 0.96) to 0.30 (0.15, 0.45) for RDs between periods 1 and 2, followed by an increase from 1.21 (1.10, 1.31) to 1.36 (1.21, 1.53) for RRs and from 0.30 (0.15, 0.45) to 0.51(0.36, 0.67) for RDs between period 2 and 3. CONCLUSION: Infant death is associated with place of residence and regional gaps have recently widened again in Korea. An in-depth investigation of the causes of regional disparities in infant mortality is required for effective governmental policies to achieve equality in infant health.

A Cell-Penetrant Peptide Disrupting the Transcription Factor CP2c Complexes Induces Cancer-Specific Synthetic Lethality.

Despite advances in precision oncology, cancer remains a global public health issue. In this report, proof-of-principle evidence is presented that a cell-penetrable peptide (ACP52C) dissociates transcription factor CP2c complexes and induces apoptosis in most CP2c oncogene-addicted cancer cells through transcription activity-independent mechanisms. CP2cs dissociated from complexes directly interact with and degrade YY1, leading to apoptosis via the MDM2-p53 pathway. The liberated CP2cs also inhibit TDP2, causing intrinsic genome-wide DNA strand breaks and subsequent catastrophic DNA damage responses. These two mechanisms are independent of cancer driver mutations but are hindered by high MDM2 p60 expression. However, resistance to ACP52C mediated by MDM2 p60 can be sensitized by CASP2 inhibition. Additionally, derivatives of ACP52C conjugated with fatty acid alone or with a CASP2 inhibiting peptide show improved pharmacokinetics and reduced cancer burden, even in ACP52C-resistant cancers. This study enhances the understanding of ACP52C-induced cancer-specific apoptosis induction and supports the use of ACP52C in anticancer drug development.

Diagnostic performance of synthetic relaxometry for predicting neurodevelopmental outcomes in premature infants: a feasibility study.

OBJECTIVES: To investigate the predictability of synthetic relaxometry for neurodevelopmental outcomes in premature infants and to evaluate whether a combination of relaxation times with clinical variables or qualitative MRI abnormalities improves the predictive performance. METHODS: This retrospective study included 33 premature infants scanned with synthetic MRI near or at term equivalent age. Based on neurodevelopmental assessments at 18-24 months of corrected age, infants were classified into two groups (no/mild disability [n = 23] vs. moderate/severe disability [n = 10]). Clinical and MRI characteristics associated with moderate/severe disability were explored, and combined models incorporating independent predictors were established. Ultimately, the predictability of relaxation times, clinical variables, MRI findings, and a combination of the two were evaluated and compared. The models were internally validated using bootstrap resampling. RESULTS: Prolonged T1-frontal/parietal and T2-parietal periventricular white matter (PVWM), moderate-to-severe white matter abnormality, and bronchopulmonary dysplasia were significantly associated with moderate/severe disability. The overall predictive performance of each T1-frontal/-parietal PVWM model was comparable to that of individual MRI finding and clinical models (AUC = 0.71 and 0.76 vs. 0.73 vs. 0.83, respectively; p > 0.27). The combination of clinical variables and T1-parietal PVWM achieved an AUC of 0.94, sensitivity of 90%, and specificity of 91.3%, outperforming the clinical model alone (p = 0.049). The combination of MRI finding and T1-frontal PVWM yielded AUC of 0.86, marginally outperforming the MRI finding model (p = 0.09). Bootstrap resampling showed that the models were valid. CONCLUSIONS: It is feasible to predict adverse outcomes in premature infants by using early synthetic relaxometry. Combining relaxation time with clinical variables or MRI finding improved prediction. CLINICAL RELEVANCE STATEMENT: Synthetic relaxometry performed during the neonatal period may serve as a biomarker for predicting adverse neurodevelopmental outcomes in premature infants. KEY POINTS: • Synthetic relaxometry based on T1 relaxation time of parietal periventricular white matter showed acceptable performance in predicting adverse outcome with an AUC of 0.76 and an accuracy of 78.8%. • The combination of relaxation time with clinical variables and/or structural MRI abnormalities improved predictive performance of adverse outcomes. • Synthetic relaxometry performed during the neonatal period helps predict adverse neurodevelopmental outcome in premature infants.

Early Discontinuation of Levothyroxine Treatment Is Safe and Feasible in Extremely Low Birth Weight Infants with Delayed Hyperthyrotropinemia.

BACKGROUND: While recent pieces of evidence suggest that discontinuation of levothyroxine replacement therapy (LRT) earlier than the current guidelines of 3 years is possible, the optimal duration of LRT for delayed hyperthyrotropinemia in extremely low birth weight infants (ELBWIs) remains unknown. OBJECTIVE: This study aimed to investigate the feasibility of early discontinuation of LRT for delayed hyperthyrotropinemia in ELBWIs. METHODS: The medical records of 92 ELBWIs who had shown delayed hyperthyrotropinemia, defined as a delayed rise in thyroid-stimulating hormone (TSH) levels to >20 µIU/mL after initial normal TSH level, were retrospectively reviewed to determine whether the duration of LRT affects the short-term outcomes at discharge from neonatal intensive care unit (NICU) and the long-term outcomes at the corrected age (CA) of 2 years. The infants were grouped into: no LRT required group (n = 21), short-term LRT given until the time of NICU discharge - 90 ± 64 (13-211) days group (n = 36), and long-term LRT given - 749 ± 333 (339-1,967) days group (n = 35). RESULTS: While mortality in the no LRT required group was significantly higher than that in the long-term LRT group, no significant differences were observed in short-term outcomes at discharge from NICU and long-term growth and neurodevelopmental outcomes at CA of 2 years between the short- and long-term LRT groups. CONCLUSIONS: Termination of LRT at around the time of discharge from NICU in well, clinically stable ELBWIs who have delayed hyperthyrotropinemia appears to be safe and feasible and avoids the risk of overtreatment.

Rational discovery of antimetastatic agents targeting the intrinsically disordered region of MBD2.

Although intrinsically disordered protein regions (IDPRs) are commonly engaged in promiscuous protein-protein interactions (PPIs), using them as drug targets is challenging due to their extreme structural flexibility. We report a rational discovery of inhibitors targeting an IDPR of MBD2 that undergoes disorder-to-order transition upon PPI and is critical for the regulation of the Mi-2/NuRD chromatin remodeling complex (CRC). Computational biology was essential for identifying target site, searching for promising leads, and assessing their binding feasibility and off-target probability. Molecular action of selected leads inhibiting the targeted PPI of MBD2 was validated in vitro and in cell, followed by confirming their inhibitory effects on the epithelial-mesenchymal transition of various cancer cells. Identified lead compounds appeared to potently inhibit cancer metastasis in a murine xenograft tumor model. These results constitute a pioneering example of rationally discovered IDPR-targeting agents and suggest Mi-2/NuRD CRC and/or MBD2 as a promising target for treating cancer metastasis.

Development of a MEL Cell-Derived Allograft Mouse Model for Cancer Research.

Murine erythroleukemia (MEL) cells are often employed as a model to dissect mechanisms of erythropoiesis and erythroleukemia in vitro. Here, an allograft model using MEL cells resulting in splenomegaly was established to develop a diagnostic model for isolation/quantification of metastatic cells, anti-cancer drug screening, and evaluation of the tumorigenic or metastatic potentials of molecules in vivo. In this animal model, circulating MEL cells from the blood stream were successfully isolated and quantified with an additional in vitro cultivation step. In terms of the molecular-pathological analysis, we were able to successfully evaluate the functional discrimination between methyl-CpG-binding domain 2 (Mbd2) and p66α in erythroid differentiation, and tumorigenic potential in spleen and blood stream of allograft model mice. In addition, we found that the number of circulating MEL cells in anti-cancer drug-treated mice was dose-dependently decreased. Our data demonstrate that the newly established allograft model is useful to dissect erythroleukemia pathologies and non-invasively provides valuable means for isolation of metastatic cells, screening of anti-cancer drugs, and evaluation of the tumorigenic potentials.

Erratum: Correction of Error in Result: Comparison of Follow-up Courses after Discharge from Neonatal Intensive Care Unit between Very Low Birth Weight Infants with and without Home Oxygen.

[This corrects the article on p. 1295 in vol. 32, PMID: 28665066.].

Mbd2-CP2c loop drives adult-type globin gene expression and definitive erythropoiesis.

During hematopoiesis, red blood cells originate from the hematopoietic stem cell reservoir. Although the regulation of erythropoiesis and globin expression has been intensively investigated, the underlining mechanisms are not fully understood, including the interplay between transcription factors and epigenetic factors. Here, we uncover that the Mbd2-free NuRD chromatin remodeling complex potentiates erythroid differentiation of proerythroblasts via managing functions of the CP2c complexes. We found that both Mbd2 and Mbd3 expression is downregulated during differentiation of MEL cells in vitro and in normal erythropoiesis in mouse bone marrow, and Mbd2 downregulation is crucial for erythropoiesis. In uninduced MEL cells, the Mbd2-NuRD complex is recruited to the promoter via Gata1/Fog1, and, via direct binding through p66α, it acts as a transcriptional inhibitor of the CP2c complexes, preventing their DNA binding and promoting degradation of the CP2c family proteins to suppress globin gene expression. Conversely, during erythropoiesis in vitro and in vivo, the Mbd2-free NuRD does not dissociate from the chromatin and acts as a transcriptional coactivator aiding the recruitment of the CP2c complexes to chromatin, and thereby leading to the induction of the active hemoglobin synthesis and erythroid differentiation. Our study highlights the regulation of erythroid differentiation by the Mbd2-CP2c loop.

Prolongation of liver-specific function for primary hepatocytes maintenance in 3D printed architectures.

Isolated primary hepatocytes from the liver are very similar to in vivo native liver hepatocytes, but they have the disadvantage of a limited lifespan in 2D culture. Although a sandwich culture and 3D organoids with mesenchymal stem cells (MSCs) as an attractive assistant cell source to extend lifespan can be used, it cannot fully reproduce the in vivo architecture. Moreover, long-term 3D culture leads to cell death because of hypoxic stress. Therefore, to overcome the drawback of 2D and 3D organoids, we try to use a 3D printing technique using alginate hydrogels with primary hepatocytes and MSCs. The viability of isolated hepatocytes was more than 90%, and the cells remained alive for 7 days without morphological changes in the 3D hepatic architecture with MSCs. Compared to a 2D system, the expression level of functional hepatic genes and proteins was higher for up to 7 days in the 3D hepatic architecture. These results suggest that both the 3D bio-printing technique and paracrine molecules secreted by MSCs supported long-term culture of hepatocytes without morphological changes. Thus, this technique allows for widespread expansion of cells while forming multicellular aggregates, may be applied to drug screening and could be an efficient method for developing an artificial liver.

Three-Dimensional Bioprinting of Hepatic Structures with Directly Converted Hepatocyte-Like Cells.

Three-dimensional (3D) bioprinting technology is a promising new technology in the field of bioartificial organ generation with regard to overcoming the limitations of organ supply. The cell source for bioprinting is very important. Here, we generated 3D hepatic scaffold with mouse-induced hepatocyte-like cells (miHeps), and investigated whether their function was improved after transplantation in vivo. To generate miHeps, mouse embryonic fibroblasts (MEFs) were transformed with pMX retroviruses individually expressing hepatic transcription factors Hnf4a and Foxa3. After 8-10 days, MEFs formed rapidly growing hepatocyte-like colonies. For 3D bioprinting, miHeps were mixed with a 3% alginate hydrogel and extruded by nozzle pressure. After 7 days, they were transplanted into the omentum of Jo2-treated NOD Scid gamma (NSG) mice as a liver damage model. Real-time polymerase chain reaction and immunofluorescence analyses were conducted to evaluate hepatic function. The 3D bioprinted hepatic scaffold (25 × 25 mm) expressed Albumin, and ASGR1 and HNF4a expression gradually increased for 28 days in vitro. When transplanted in vivo, the cells in the hepatic scaffold grew more and exhibited higher Albumin expression than in vitro scaffold. Therefore, combining 3D bioprinting with direct conversion technology appears to be an effective option for liver therapy.

Comparison of Follow-up Courses after Discharge from Neonatal Intensive Care Unit between Very Low Birth Weight Infants with and without Home Oxygen.

In order to investigate the clinical impact of home oxygen use for care of premature infants, we compared the follow-up courses after neonatal intensive care unit (NICU) discharge between very low birth weight infants (VLBWIs) with and without home oxygen. We retrospectively identified 1,232 VLBWIs born at 22 to 32 weeks of gestation, discharged from the NICU of 43 hospitals in Korea between April 2009 and March 2010, and followed them up until April 2011. Clinical outcomes, medical service uses, and readmission and death rates during follow-up after the NICU discharge were compared between VLBWIs with (HO, n = 167) and those without (CON, n = 1,056) home oxygen at discharge. The HO infants comprised 13.7% of the total VLBWIs with significant institutional variations and showed a lower gestational age (GA) and birth weight than the CON infants. The HO infants had more frequent regular pediatric outpatient clinic visits (12.7 ± 7.5 vs. 9.5 ± 6.6; P < 0.010) and emergency center visits related to respiratory problems (2.5 ± 2.2 vs. 1.8 ± 1.4; P < 0.010) than the CON infants. The HO infants also had significantly increased readmission (adjusted hazard ratio [HR], 1.60; 95% confidence interval [CI], 1.25-2.04) and death risks (adjusted HR, 7.40; 95% CI, 2.06-26.50) during up to 2 years following the NICU discharge. These increased readmission and death risks in the HO infants were not related to their prematurity degree. In conclusion, home oxygen use after discharge increases the risks for healthcare utilization, readmission, and death after NICU discharge in VLBWIs, regardless of GA, requiring more careful health care monitoring during their follow-up.

Diagnostic and prognostic relevance of CP2c and YY1 expression in hepatocellular carcinoma.

Recent studies have demonstrated an oncogenic role of the transcription factor (TF) CP2c in hepatocellular carcinoma (HCC) based on a strong correlation between CP2c expression, tumor grade, and aggressiveness. We recently found that CP2c directly interacts with another TF, YY1, which is also overexpressed in multiple cancers, including HCC. To evaluate if these proteins are co-regulated in carcinogenesis, we analyzed the expression of CP2c and YY1 in HCC (n = 136) tissues and examined the correlation between their expression and clinicopathological characteristics of HCC. Receiver operating characteristic analysis exhibited the validity of CP2c and nuclear YY1 expression as a diagnostic factor in HCC tissues. High expression of CP2c was significantly correlated with patient age, and higher histological grade, American Joint Committee on Cancer (AJCC) stage, and small and large vessel invasion in HCC tissues, whereas high expression of nuclear YY1 was significantly associated with higher AJCC stage and small vessel invasion. In univariate and multivariate analyses, high expression of CP2c was significantly correlated with disease free survival (DFS), indicating that CP2c expression is an independent prognostic factor for DFS in HCC patients. Patients with high expression of both CP2c and nuclear YY1 usually had a shorter median survival time and worse DFS prognosis than other patients, suggesting that combined detection of CP2c and nuclear YY1 is a useful prognostic marker in HCC patients.

Three-dimensional (3D) printing of mouse primary hepatocytes to generate 3D hepatic structure.

PURPOSE: The major problem in producing artificial livers is that primary hepatocytes cannot be cultured for many days. Recently, 3-dimensional (3D) printing technology draws attention and this technology regarded as a useful tool for current cell biology. By using the 3D bio-printing, these problems can be resolved. METHODS: To generate 3D bio-printed structures (25 mm × 25 mm), cells-alginate constructs were fabricated by 3D bio-printing system. Mouse primary hepatocytes were isolated from the livers of 6-8 weeks old mice by a 2-step collagenase method. Samples of 4 × 107 hepatocytes with 80%-90% viability were printed with 3% alginate solution, and cultured with well-defined culture medium for primary hepatocytes. To confirm functional ability of hepatocytes cultured on 3D alginate scaffold, we conducted quantitative real-time polymerase chain reaction and immunofluorescence with hepatic marker genes. RESULTS: Isolated primary hepatocytes were printed with alginate. The 3D printed hepatocytes remained alive for 14 days. Gene expression levels of Albumin, HNF-4α and Foxa3 were gradually increased in the 3D structures. Immunofluorescence analysis showed that the primary hepatocytes produced hepatic-specific proteins over the same period of time. CONCLUSION: Our research indicates that 3D bio-printing technique can be used for long-term culture of primary hepatocytes. It can therefore be used for drug screening and as a potential method of producing artificial livers.

Reciprocal localization of transcription factors YY1 and CP2c in spermatogonial stem cells and their putative roles during spermatogenesis.

Maintaining stemness and permitting differentiation mediated by combinations of transcription factors (TFs) are key aspects of mammalian spermatogenesis. It has been established that yin yang 1 (YY1), a target factor of mammalian polycomb repressive complex 2 (PRC2) and a regulator of stemness, is involved in the stable maintenance of prophase stage spermatocytes. Recently, we have demonstrated that the TF CP2c partners with YY1 in some cells to antagonistically regulate the other protein's function. To date, the functional roles of YY1 and CP2c in spermatogonial stem cells and their derived germ cells remain unclear. Here, we investigated the expression of YY1 and CP2c in mouse gonocytes and germ cells using tissue immunohistochemical and immunofluorescence analyses. At E14.5, both YY1 and CP2c were stained in gonocytes and Sertoli cells in testicular cords, showing different proportion and density of immunoreactivity. However, in adult testes, YY1 was localized in the nuclei of spermatogonial stem cells and spermatocytes, but not in spermatozoa. It was also detected in spermatogonia and spermatids in a stage-specific manner during spermatogenic cycle. CP2c could be detected mostly in the cytoplasm of spermatocytes but not at all in spermatogonial stem cells, indicating mutually exclusive expression of CP2c and YY1. Interestingly, however, CP2c was stained in the cytoplasm and nucleus of spermatogonia at elongation and release stages, and co-localized with YY1 in the nucleus at grouping, maturation, and releasing stages. Neither YY1 nor CP2c was expressed in spermatozoa. Our data indicate that YY1 strongly localizes in the spermatogonial stem cells and co-localizes heterogeneously with CP2c to permit spermatogenesis, and also suggest that YY1 is essential for stemness of spermatogonial stem cells (SCs) whereas CP2c is critical for the commitment of spermatogonia and during the progression of spermatogonia to spermatids. This evaluation expands our understanding of the molecular mechanism of spermatogonia formation as well as spermatogenesis in general.

The regulation of glucose-6-phosphatase and phosphoenolpyruvate carboxykinase by autophagy in low-glycolytic hepatocellular carcinoma cells.

The glycolytic phenotype is a dominant metabolic phenomenon in cancer and is reflected in becoming aggressive. Certain hepatocellular carcinoma lack increased glycolysis and prefer to uptake acetate than glucose for metabolism. Autophagy plays a role in preserving energies and nutrients when there is limited external nutrient supply and maintains glucose level of blood though supporting gluconeogenesis in the liver. As the role of autophagy and gluconeogenesis in HCC following the glycolic activity was not clear, we cultured HCC cells with different glycolytic levels in Hank's balanced salt solution (HBSS) to induce autophagy and conducted the activity of gluconeogenesis. Both autophagy and gluconeogenesis were induced in low glycolytic HCC cells (HepG2). In glycolytic Hep3B cells, only autophagy without gluconeogenesis was induced upon starvation. When autophagy was blocked, the level of glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (PEPCK) was reduced in HepG2 cells and not in Hep3B. Altogether, we investigated contribution of hepatic gluconeogenesis to the metabolic phenotype of HCC cells and the role of autophagy as a potential mechanism regulating gluconeogenesis in low glycolytic HCC.

An intracellular antifreeze protein from an Antarctic microalga that responds to various environmental stresses.

The structure and function of the Antarctic marine diatom Chaetoceros neogracile antifreeze protein (Cn-AFP), as well as its expression levels and characteristics of the ice-binding site, were analyzed in the present study. In silico analysis revealed that the Cn-AFP promoter contains both light- and temperature-responsive elements. Northern and Western blot analyses demonstrated that both Cn-AFP transcript and protein expression were strongly and rapidly stimulated by freezing, as well as temperature and high light stress. Immunogold labeling revealed that Cn-AFP is preferentially localized to the intracellular space near the chloroplast membrane. Recombinant Cn-AFP had clear antifreeze activity. Protein-folding simulation was used to predict the putative ice-binding sites in Cn-AFP, and site-directed mutagenesis of the Cn-AFP b-face confirmed their identification.