[Construction and effect evaluation of tuberculosis information platform in Ningxia].

Objective: To explore the value of information technology in management of tuberculosis patients. Methods: The data comes from the case reports of Ningxia tuberculosis patients in the national tuberculosis special report system from March to December 2019 and the data of Ningxia tuberculosis information management platform. Using the computer Information and communication technologies and the Internet platform, unified data collection and data interface standards,Hospital Information System(HIS) of 18 (90%)TB specific Hospitals in Ningxia 22 counties are linked together, forming a closed loop management System, to realize Information interconnection, compare the patients' diagnostic rate before and after the application, and standardize treatment differences. Results: The system automatically collected and uploaded valid data for 2 918 times, and the consistency rate of uploaded data was 94.8%.The clinical diagnostic rate was 18.23% (1 154/6 332) before and 39.1% (914/2 335) after using the AI imaging screening subsystem (χ²=235.56, P<0.001).The regular medication rate of patients after system use was 86.89% (510/587), which was higher than that before [73.72% (544/738)] (χ²=3.94, P<0.05).The cure rate of etiologically positive tuberculosis patients (2019) was 86.03% (788/916), which was higher than that of 83.45% (1 008/1 208) before the use of the system (2018) (χ²=435.43, P<0.001). Conclusion: The tuberculosis information management platform in Ningxia has achieved tuberculosis information interconnection, significantly improving the standard diagnosis, treatment and management level of tuberculosis.

Search for a Variation of the Fine Structure Constant around the Supermassive Black Hole in Our Galactic Center.

Searching for space-time variations of the constants of Nature is a promising way to search for new physics beyond general relativity and the standard model motivated by unification theories and models of dark matter and dark energy. We propose a new way to search for a variation of the fine-structure constant using measurements of late-type evolved giant stars from the S star cluster orbiting the supermassive black hole in our Galactic Center. A measurement of the difference between distinct absorption lines (with different sensitivity to the fine structure constant) from a star leads to a direct estimate of a variation of the fine structure constant between the star's location and Earth. Using spectroscopic measurements of five stars, we obtain a constraint on the relative variation of the fine structure constant below 10^{-5}. This is the first time a varying constant of nature is searched for around a black hole and in a high gravitational potential. This analysis shows new ways the monitoring of stars in the Galactic Center can be used to probe fundamental physics.

Microrheology and Spatial Heterogeneity of Staphylococcus aureus Biofilms Modulated by Hydrodynamic Shear and Biofilm-Degrading Enzymes.

Particle tracking microrheology was used to investigate the viscoelasticity of Staphylococcus aureus biofilms grown in microfluidic cells at various flow rates and when subjected to biofilm-degrading enzymes. Biofilm viscoelasticity was found to harden as a function of shear rate but soften with increasing height away from the attachment surface in good agreement with previous bulk results. Ripley's K-function was used to quantify the spatial distribution of the bacteria within the biofilm. For all conditions, biofilms would cluster as a function of height during growth. The effects of proteinase K and DNase-1 on the viscoelasticity of biofilms were also investigated. Proteinase K caused an order of magnitude change in the compliances, softening the biofilms. However, DNase-1 was found to have no significant effects over the first 6 h of development, indicating that DNA is less important in biofilm maintenance during the initial stages of growth. Our results demonstrate that during the preliminary stages of Staphylococcus aureus biofilm development, column-like structures with a vertical gradient of viscoelasticity are established and modulated by the hydrodynamic shear caused by fluid flow in the surrounding environment. An understanding of these mechanical properties will provide more accurate insights for removal strategies of early-stage biofilms.

Testing General Relativity with Stellar Orbits around the Supermassive Black Hole in Our Galactic Center.

We demonstrate that short-period stars orbiting around the supermassive black hole in our Galactic center can successfully be used to probe the gravitational theory in a strong regime. We use 19 years of observations of the two best measured short-period stars orbiting our Galactic center to constrain a hypothetical fifth force that arises in various scenarios motivated by the development of a unification theory or in some models of dark matter and dark energy. No deviation from general relativity is reported and the fifth force strength is restricted to an upper 95% confidence limit of |α|<0.016 at a length scale of λ=150 astronomical units. We also derive a 95% confidence upper limit on a linear drift of the argument of periastron of the short-period star S0-2 of |ω[over ˙]_{S0-2}|<1.6×10^{-3} rad/yr, which can be used to constrain various gravitational and astrophysical theories. This analysis provides the first fully self-consistent test of the gravitational theory using orbital dynamic in a strong gravitational regime, that of a supermassive black hole. A sensitivity analysis for future measurements is also presented.

Adsorption of Bovine Serum Albumin (BSA) at the Oil/Water Interface: A Neutron Reflection Study.

The structure of the adsorbed protein layer at the oil/water interface is essential to the understanding of the role of proteins in emulsion stabilization, and it is important to glean the mechanistic events of protein adsorption at such buried interfaces. This article reports on a novel experimental methodology for probing protein adsorption at the buried oil/water interface. Neutron reflectivity was used with a carefully selected set of isotopic contrasts to study the adsorption of bovine serum albumin (BSA) at the hexadecane/water interface, and the results were compared to those for the air/water interface. The adsorption isotherm was determined at the isoelectric point, and the results showed that a higher degree of adsorption could be achieved at the more hydrophobic interface. The adsorbed BSA molecules formed a monolayer on the aqueous side of the interface. The molecules in this layer were partially denatured by the presence of oil, and once released from the spatial constraint by the globular framework they were free to establish more favorable interactions with the hydrophobic medium. Thus, a loose layer extending toward the oil phase was clearly observed, resulting in an overall broader interface. By analogy to the air/water interface, as the concentration of BSA increased to 1.0 mg mL(-1) a secondary layer extending toward the aqueous phase was observed, possibly resulting from the steric repulsion upon the saturation of the primary monolayer. Results clearly indicate a more compact arrangement of molecules at the oil/water interface: this must be caused by the loss of the globular structure as a consequence of the denaturing action of the hexadecane.

Effect of polymorphisms of vascular endothelial growth factor on prognosis in osteosarcoma patients.

We investigated the association between vascular endothelial growth factor (VEGF) gene +1612G/A, -634C/G, and +936G/C and the clinical outcome of osteosarcoma. Genomic DNA was isolated from blood samples, and 3 VEGF gene polymorphisms (+1612G/A, -634C/G, and +936G/C) were analyzed using polymerase chain reaction-restriction fragment length polymorphism. Of the 194 patients, 82 patients (42.27%) showed a good response to chemotherapy, while 73 (37.63%) died during the follow-up period. When comparing good and poor responders, we observed no significant association between the VEGF +1612G/A, -634G/C, and +936T/C polymorphisms and clinical outcome of osteosarcoma patients. According to Cox regression analysis, the VEGF +1612A/G, -634G/C, and +936T/C polymorphisms did not statistically significantly increase the risk of overall survival of patients with osteosarcoma. This study showed that VEGF +1612A/G, -634G/C, and +936T/C polymorphisms were not related to the response to chemotherapy and clinical outcome of osteosarcoma patients.

Factors influencing the occurrence of hyperuricemia and poor cardiac and renal outcomes in chronic kidney disease.

OBJECTIVE: The aim of our study was to analyze the factors influencing the occurrence of hyperuricemia and poor cardiac and renal outcomes in chronic kidney disease (CKD). PATIENTS AND METHODS: One hundred and sixteen patients with CKD admitted to our hospital from January 2022 to September 2022 were picked as the subjects. Fasting venous blood of these subjects was collected to value the serum uric acid (SUA) levels on an automatic biochemical analyzer. Patients were then grouped as the CKD-only group (n=80) and hyperuricemia group (n=36), according to the SUA results, or the good prognosis group (n=88) and poor prognosis group (n=28), according to the presence of cardiovascular diseases. The changes in laboratory indexes and clinical data were analyzed and compared. Multivariate logistic regression analysis was used to analyze the risk factors for combined hyperuricemia and the risk factors for poor cardiac and renal outcomes in patients with CKD. The correlation between SUA level and cardiac and renal indexes was analyzed by Pearson analysis. RESULTS: Patients in the CKD hyperuricemia group had markedly higher content of systolic blood pressure (SBP), diastolic blood pressure (DBP), B-type natriuretic peptide (BNP), urinary retinol-binding protein (RBP), urinary N-acetyl-ß-D glucosidase (NAG), much higher proportion of heart failure episodes history, and much lower content of total cholesterol (TC), albumin (Alb), hemoglobin (Hb), urinary α1-microglobulin (α1-MG), and glomerular filtration rate (eGFR) than the CKD-only group (p < 0.05). SUA, BNP, SBP, and history of heart failure episodes were independent risk factors for combined hyperuricemia in CKD patients (p < 0.05). Besides, eGFR, albumin, and hemoglobin were independent protective factors for combined hyperuricemia in CKD patients (p < 0.05). Compared with the good prognosis group, the content of BNP, SBP, DBP, urinary RBP, urinary NAG, and SUA was much higher, the proportion of heart failure episodes history was obviously higher, and the levels of Alb, Hb, TC, eGFR, and urinary α1-MG were sharply lower in the poor prognosis group (p < 0.05). SUA, BNP, SBP, and history of heart failure episodes were independent risk factors for poor cardiac and renal outcomes (p < 0.05), and eGFR was an independent protective factor for poor cardiac and renal outcomes in patients with CKD (p < 0.05). The SUA level in CKD patients was positively correlated with BNP and SBP (r=0.463, 0.215, p < 0.05), but negatively correlated with eGFR (r=0.463, 0.215, p < 0.05). CONCLUSIONS: The serum SUA level was elevated with the aggravation of the CKD stage. High serum SUA level is a risk factor for the development of hyperuricemia and poor cardio-renal outcomes in CKD patients, suggesting that early monitoring of changes in SUA levels may help assess the risk of cardio-renal outcomes in CKD patients.

Electronics of peptide- and protein-based biomaterials.

Biologically inspired peptide- and protein-based materials are at the forefront of organic bioelectronics research due to their inherent conduction properties and excellent biocompatibility. Peptides have the advantages of structural simplicity and ease of synthesis providing credible prospects for mass production, whereas naturally expressed proteins offer inspiration with many examples of high performance evolutionary optimised bioelectronics properties. We review recent advances in the fundamental conduction mechanisms, experimental techniques and exemplar applications for the bioelectronics of self-assembling peptides and proteins. Diverse charge transfer processes, such as tunnelling, hopping and coupled transfer, are found in naturally occurring biological systems with peptides and proteins as the predominant building blocks to enable conduction in biology. Both theory and experiments allow detailed investigation of bioelectronic properties in order to design functionalized peptide- and protein-based biomaterials, e.g. to create biocompatible aqueous electrodes. We also highlight the design of bioelectronics devices based on peptides/proteins including field-effect transistors, piezoelectric energy harvesters and optoelectronics.

Health-related quality of life predicts hospital admission within 1 year in people with diabetes: a nationwide study from Taiwan.

AIMS: To investigate whether health-related quality of life (HRQOL) predicts hospital admission in a nationally representative sample of adults with diabetes. METHODS: We conducted a prospective study on persons aged > or = 18 years with self-reported physician-diagnosed diabetes (n = 797) who participated in the National Health Interview Survey in Taiwan, 2001. Of these potential participants, 674 provided consent for data linkage and were successfully linked to the National Health Insurance claims data. We analysed the associations between the Short Form 36 (SF-36) subscales and summaries and the occurrence of hospital admission for any cause during 2002. RESULTS: Approximately 23% of participants with diabetes had at least one hospital admission during 2002. After adjusting for demographic characteristics, co-morbidities and diabetics-related attributes, those who had been admitted to hospital had significantly poorer mean scores on each of the physical dimensions, physical components summary (PCS) and social functioning domain of the SF-36 at baseline. In logistic regression models, poorer scores on the PCS [odds ratio (OR) = 1.80; 95% confidence interval (CI) = (1.14-2.86)], duration of diabetes > or = 10 years [OR = 2.10; 95% CI = (1.14-3.89)] and the presence of heart disease [OR = 1.63; 95% CI = (1.01-2.63)] were significantly associated with an increased risk of hospital admission. CONCLUSION: In people with diabetes, poorer scores on the PCS of the SF-36 at baseline may provide additional information for assessment of hospital admission risk, independent of other measures of health outcomes.

pH-responsive nanoaggregation of diblock phosphorylcholine copolymers.

We have characterized three diblock copolymers bearing zwitterionic phosphorylcholine and weak tertiary amine groups, namely, poly[((2-(methacryloyloxy)ethyl)phosphorylcholine)30- block-(2-(dimethylamino)ethyl methacrylate)60] (denoted as MPC30-DMA60, Mn=18,000), poly[((2-(methacryloyloxy)ethyl)phosphorylcholine)30- block-(2-(diethylamino)ethyl methacrylate)60) (denoted as MPC30-DEA60, Mn=20,000), and poly[((2-(methacryloyloxy)ethyl)phosphorylcholine)30- block-(2-(diisopropylamino)ethyl methacrylate)60) (denoted as MPC30-DPA60, Mn=21,000), by studying their surface tension and solution aggregation through a combined approach of surface tension measurement, dynamic light scattering, and small-angle neutron scattering. Our results show that larger tertiary amine substituents lead to an increasing tendency to form micellar aggregates, which is consistent with the increasing copolymer hydrophobicity. Thus, MPC30-DMA60 did not aggregate under the experimental conditions studied. The free chains exist in the form of thin cylinders, whose length decreases with copolymer concentration and solution temperature but increases with solution pH. The diameters of the MPC30-DMA60 cylinders remained almost constant at around 30 A under all the conditions studied. At the lower copolymer concentration of 0.5 wt %, the cylindrical lengths correspond to the persistence length of the copolymer backbone and are close to its full length, indicating a rather high rigidity. Further data analysis showed that, at the two higher concentrations of 2 and 4 wt %, the phosphorylcholine and amine blocks associate, inducing bending of the copolymer backbone. One backbone kink was required to satisfy all the constraints, including the dry volume of the copolymer. MPC30-DEA60 showed a similar trend of pH- and concentration-dependent conformational responses for the free copolymer, but in addition micellar aggregation occurred at pH 9. In contrast, MPC30-DPA60 exhibited significantly reduced solubility associated with strong aggregation, which is consistent with it being the most hydrophobic copolymer in the series.

LncRNA HOTAIR participates in the development and progression of adrenocortical carcinoma via regulating cell cycle.

OBJECTIVE: To explore the role of HOTAIR in the pathogenesis of adrenocortical carcinoma (ACC) and its underlying mechanism. PATIENTS AND METHODS: Differentially expressed lncRNA (HOTAIR) in ACC was screened out from the GEO database. The survival analysis and ROC curve were performed according to HOTAIR expressions in ACC patients. The correlation between HOTAIR expression and clinical information of ACC patients was analyzed by chi-square test. The univariate and multivariate COX regression analysis was carried out to analyze the relationship between HOTAIR expression, disease-free survival (DFS) and overall survival (OS) of ACC patients. We then detected HOTAIR expression in 77 ACC tissues and 30 normal tissues by qRT-PCR (quantitative Real-time polymerase chain reaction). ACC cell lines were further screened out for the following in vitro experiments. After altering HOTAIR expression in ACC cells by plasmid transfection, proliferation and cell cycle were detected by Cell Counting Kit-8 (CCK-8) and colony formation assay, respectively. Finally, Western blot was utilized to detect expressions of cell cycle-related genes in ACC cells. RESULTS: HOTAIR was overexpressed in ACC tissues than that of normal tissues. HOTAIR expression was remarkably increased in ACC with T3 and T4 stage than that of T1 and T2 stage. Moreover, HOTAIR expression was remarkably increased in ACC with stage III and IV than that of stage I and II. HOTAIR was an independent prognostic factor for DFS and OS of ACC patients. For in vitro experiments, inhibited proliferation and arrested cell cycle were observed in H295R cells transfected with si-HOTAIR. Opposite results were obtained after SW-13 cells were transfected with HOTAIR overexpression plasmid. Furthermore, expressions of cell cycle-related genes, including Cyclin D1, p-Rb and p-GSK3ß were remarkably decreased after HOTAIR knockdown. CONCLUSIONS: We demonstrated for the first time that HOTAIR is overexpressed in ACC and is a prognostic risk factor in ACC patients. HOTAIR participates in the development and progression of ACC via shortening cell cycle and promoting proliferation of ACC cells.

FOG-2, a heart- and brain-enriched cofactor for GATA transcription factors.

Members of the GATA family of zinc finger transcription factors have been shown to play important roles in the control of gene expression in a variety of cell types. GATA-1, -2, and -3 are expressed primarily in hematopoietic cell lineages and are required for proliferation and differentiation of multiple hematopoietic cell types, whereas GATA-4, -5, and -6 are expressed in the heart, where they activate cardiac muscle structural genes. Friend of GATA-1 (FOG) is a multitype zinc finger protein that interacts with GATA-1 and serves as a cofactor for GATA-1-mediated transcription. FOG is coexpressed with GATA-1 in developing erythroid and megakaryocyte cell lineages and cooperates with GATA-1 to control erythropoiesis. We describe a novel FOG-related factor, FOG-2, that is expressed predominantly in the developing and adult heart, brain, and testis. FOG-2 interacts with GATA factors, and interaction of GATA-4 and FOG-2 results in either synergistic activation or repression of GATA-dependent cardiac promoters, depending on the specific promoter and the cell type in which they are tested. The properties of FOG-2 suggest its involvement in the control of cardiac and neural gene expression by GATA transcription factors.

Neutron reflection from a dimyristoylphosphatidylcholine monolayer adsorbed on a hydrophobised silicon support.

Neutron specular reflection has been used to study the structure of a monolayer of dimyristoylphosphatidylcholine (DMPC) deposited using the Langmuir-Blodgett technique onto a silicon oxide substrate. A self-assembled monolayer of octadecyltrichlorosilane with a deuterated alkyl chain (d-OTS) had been previously bonded onto this silicon oxide substrate which rendered it hydrophobic. In the system under study, the alkyl chains of the phospholipid were found to penetrate extensively into the d-OTS layer with the mixed chain region (d-OTS and DMPC) having a total thickness of 30.5 A. This mixed region was divided into two halves for analysis; the 'lower half' (nearest to the substrate surface) was found to comprise anchored d-OTS chains mixed with the lipid chains in the volume ratio approx. 0.60:0.35. The corresponding volume ratio in the 'upper half' of this region was determined to be approx. 0.50:0.40. The thicknesses of these regions were found to be 17.9 A (incorporating approx. 6% solvent) and 12.6 A (incorporating approx. 9% solvent) for the lower and upper halves respectively. The DMPC head groups were found to be confined to the most external layer (furthest away from the silicon substrate). This layer was found to have a thickness of 9.4 A and included a small fraction of the lipid alkyl chains with approx. 47% solvent.

Adsorption of single chain zwitterionic phosphocholine surfactants: effects of length of alkyl chain and head group linker.

The adsorption of a range of single chain zwitterionic phosphocholine surfactants (C(n)P(m)C) at the air/liquid interface has been studied by a combination of surface tension and neutron reflectivity. The critical micellar concentration (CMC) for C(n)PC (or C(n)P(2)C), where n varied from 12, 14 to 16, was found to be 0.91, 0.14, and 1.2 x 10(-2) mM respectively, and followed the same trend as observed for other zwitterionic and non-ionic surfactants. The area per molecule at the CMC, A(cmc), for C(n)PC was found to remain constant between 50 and 53 A(2), indicating that the increase in the alkyl chain length had little effect on A(cmc) at the interface. The neutron reflection measurement also showed an almost constant layer thickness (tau) of 20+/-2 A from all the alkyl chain deuterated PC surfactants (dC(n)hPC) in null reflecting water (NRW), suggesting that the alkyl chains of the surfactant responded to changes in either chain length or solution concentration by varying their angle of tilt. In contrast, increasing the length of head group linker between P and N atoms in C(12)P(m)C, where m=2, 4, to 6, resulted in a much slower decrease of CMC from 0.91, 0.7, to 0.5 mM, consistent with a different contribution to the free energy of micellization. A(cmc) for C(12)P(m)C did not vary when m was increased from 2 to 4, and this observation together with the thickness of the head group region indicated an almost perpendicular projection of the head group in C(12)P(2)C and C(12)P(4)C. A further increase in m to 6 resulted in an A(cmc) of 70 A(2). This increase in A(cmc) however did not result in any change in either the total layer thickness or the fraction of the head group region submerged in the aqueous subphase, suggesting that the head group in C(12)P(6)C was bent away from the surface normal direction. Both increase in temperature from 25 to 40 degrees C and the addition of 0.1 M NaCl had little effect on the area per molecule or the thickness of C(12)P(m)C surfactant layer, showing that the C(12)P(m)C series behaved like C(n)P(2)C series. The main conclusion from this study is that for all the C(n)P(m)C surfactants studied, change in m or n has little effect on the total thickness, the thickness of the alkyl chain or that of the head group region.

Surface adsorption of zwitterionic surfactants: n-alkyl phosphocholines characterised by surface tensiometry and neutron reflection.

The surface adsorption of n-dodecyl phosphocholine (C12PC) has been characterised by a combined measurement of surface tension and neutron reflectivity. The critical micellar concentration (CMC) was found to be 0.91 mM at 25 degrees C in pure water. At the CMC, the limiting area per molecule (A(cmc)) was found to be 52+/-3 A2 and the surface tension (gamma(cmc)) to be ca. 40.0+/-0.5 mN/m. The parallel study of chain isomer n-hexadecyl phosphocholine (C16PC) showed a decrease of the CMC to 0.012 mM and a drop of gamma(cmc) to 38.1+/-0.5 mN/m. However, A(cmc) for C16PC was found to be 54+/-3 A2, showing that increase in alkyl chain length by four methylene groups has little effect on A(cmc). The almost constant A(cmc) suggested that the limiting area per molecule was determined by the bulky PC head group. It was further found that the surface tension and related key physical parameters did not vary much with temperature, salt addition, solution pH or any combination of these, thus showing that surface adsorption and solution aggregation from PC surfactants is largely similar to the zwitterionic betaine surfactants and is distinctly different from ionic and non-ionic surfactants. The thickness of the adsorbed monolayers measured from both dC12hPC and dC16hPC was found to be 20-22 A at the CMC from neutron reflectivity. Neither A(cmc) nor layer thickness varied with alkyl chain length, indicating that as the alkyl chain length became longer it was further tilted away from the surface normal direction and the layer packing density increased. It was also observed that the thickness of the layer varied little with surfactant concentration, indicating that the average conformational orientation of the alkyl chain remained unchanged against varying surface coverage.

The reduced adsorption of lysozyme at the phosphorylcholine incorporated polymer/aqueous solution interface studied by spectroscopic ellipsometry.

Coating hydrogel polymers onto solid substrates can reduce the adsorption of proteins onto these surfaces, but the extent of the reduction in protein adsorption is strongly dependent on how the surface layer is coated. We have examined the effect of coating conditions on the structure of thin polymer films formed from a number of poly(methacrylate)-based hydrogel polymers via the dip-coating method. We show in this work how the polarity of the solvent, the speed of lifting, and the annealing temperature affect the thickness and uniformity of ultrathin phosphorylcholine (PC)-incorporated polymer films coated on the surface of native oxide on silicon and the subsequent interaction of these coated surfaces with lysozyme molecules. Our results show that the uniformity of the polymer film, and thus the smoothness of the outer film surface, influence the extent of reduction in protein adsorption. We suggest that the reduction in lysozyme adsorption is the result of a layer of PC groups on the surface of the polymer film. The improvement of the smoothness of the film results in the formation of a close-packed PC layer on the outer surface of the polymer film, leaving few defects or cavities on which protein molecules can bind.

Surfactant layers at the air/water interface: structure and composition.

The use of neutron reflectometry to study the structure and composition of surfactant layers adsorbed at the air/water interface is reviewed. A critical assessment of the results from this new technique is made by comparing them with the information available from all other techniques capable of investigating this interface.

Pollution by PCDDS and PCDFS in sediment from freshwater fish culture ponds near incineration sites for metal reclamation in Wan-Li, Taiwan, Republic of China.

Sediment samples from nine culture ponds for freshwater fish at and near waste incineration sites for metal reclamation in Wan-Li, southern Taiwan, Republic of China were analyzed for PCDDs and PCDFs by HRGC-HRMS. Four of nine samples were heavily polluted by PCDDs and PCDFs, indicating the latter to be dominant. The total TEQ concentrations of PCDDs and PCDFs in the four samples were in the range of 257 to 12200 pg/g dry weight. In addition, the levels corresponded to those of surface soil samples from the same waste combustion areas.

Identification of the types of adenovirus isolates with monoclonal antibodies.

The types of 59 isolates of adenovirus (Adv) which were isolated between winter, 1984 and spring, 1986 were identified by type-specific monoclonal antibodies (McAb) against Adv types 3 and 7. The results showed that the method could not only identify the types of Adv, but also discover the changes of their subtypes with time. The tedious neutralization test can be replaced by this simple method.