Promoting the exploration and research of hospital patent transformation through multiple channels / 中华医学科研管理杂志

Objective:The medical-related patent transformation level is relatively low in China for a long time. However, with the support and advancement of a series of national policies, domestic patents and other medical intellectual property projects are increasing year by year, and medical transformation is also receiving more and more attention. Zhongshan Hospital affiliated to Fudan University actively promotes the transformation of hospital patents through various channels, and actively explores how to carry out patent transformation and how to improve the success rate of transformation. This article aims to analyze and explore the feasibility of multi-channel promotion of patents, and to open up a new path for the use of new media to promote patent conversion.Methods:Through Excel analysis of " Zhongshan Patent Hero Post" WeChat public account and official Weibo platform weekly page views data and patent salon project data, as well as the patent application and transformation of Zhongshan Hospital in the past 6 years, and analyze the effect of patent promotion.Results:Through multiple channels and various links of effective promotion methods, the patent application and conversion situation of Zhongshan Hospital has doubled in the past 6 years.Conclusions:The hospital will further actively explore the patent application and transformation process, the full-cycle promotion method of each link, and provide a transfer and transformation consulting docking and promotion platform for the scientific research results or patent technologies related to hospitals and enterprises, and help the transformation of medical patents.

Preparation of human monoclonal anti-C cell line from peripheral blood B lymphocytes of D--donor / 中国输血杂志

【Objective】 To establish human hybridoma cell lines, secreting monoclonal antibody against antigens of Rh blood system, from a donor with rare D--phenotype. 【Methods】 Peripheral blood B lymphocytes of an O type female donor, lacking C/c/E/e antigens on her erythrocyte, were transformed with Epstein-Barr virus (EBVs). EBVs were harvested from the cultural supernatant of B95-8 cells. The transformed lymphoblastoid cell line (LCL) secreting antibodies to C antigens were picked up and then hybridized with the myeloma SHM-D33 using electric fusion technique. Hybridoma cells were selected by HATD-Ouabain(HOTD)(Hypoxantine, Aminopterin, Thymidine, 2-Deoxycytide, and Ouabain)culture medium, microplate antibody screening and limited dilution subcloning. The monoclonal antibody was assayed by serological test and was confirmed by flow cytometry (FCM). 【Results】 From the cultural supernatant of D--peripheral blood transformed B lymphocytes, 3A6-C6, which agglutinated with R1R1(DCe/DCe)O-type RBCs but not with R2R2(DcE/DcE)O-type RBCs, was screened and preliminarily identified as anti-C. We established a hybridoma cell line secreting anti-C immunoglobulin M from B cells of D--individual successfully after hybridization with SHM-D33 myeloma cells. 【Conclusion】 The study had laid the groundwork for future research and development of human monoclonal antibodies against Rh antigens of RBC in future for diagnosis and screening purpose.

Application of MALDI-TOF MS in clinical difficult blood group typing / 中国输血杂志

【Objective】 To explore the application of matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) in the genotyping of difficult blood typing samples, and to provide evidence for clinical blood transfusion. 【Methods】 Three ambiguous blood group samples, submitted to Shanghai Blood Center by Shanghai regional hospitals, were studied, of which Sample1 included the proband and his parents. Serological methods were used to perform blood group typing, direct antibody test, unexpected antibody screening and identification test. Blood group genotyping was performed by using the MALDI-TOF MS detection systeme stablished in our laboratory. Sanger sequencing was used to confirm gene mutation sites, and serological or flow methods were used to verify specific samples′ phenotype. 【Results】 Serological results indicated the existence of antibodies against high frequency antigens in sample 1 (including proband and her mother), 2 and 3. The genotyping results of MALDI-TOF MS showed that the proband of sample 1 was Di(a+ b+ ), her father was Di(a-b+ ), her mother was Di(a+ b-), sample 2 was p, and sample 3 was Jr(a-). Sequencing results of three samples were consistent with mass spectrometry typing results. Serological results showed that sample 2 had a p phenotype. The flow cytometry results suggested that sample 3 had a Jr(a-) phenotype. 【Conclusion】 For the first time, we applied MALDI-TOF MS technology to blood type genotyping of ambiguous clinical samples in China. Compared with other genotyping methods such as PCR-SSP, MALDI-TOF MS has the advantages of rapid detection, high throughput and high specificity, which would contribute to identification of difficult blood typing samples in the future, as well as rare blood group screening.

β-Lactam antibiotics promoting aging and clearance of RBCs would deteriorate the DIIHA / 中国输血杂志

【Objective】 To analyze the influence of β-lactam antibiotics on RBC aging and clearance by detecting various indicators of aging and clearance on RBCs, as well as the differences in phagocytosis for erythrocytes before and after drugs treated in vitro. 【Methods】 RBCs were treated by β-lactam antibiotics, including Penicillin, Cefepime, Cefoperazone and Ceftazidime, and the changing of phosphatidylserine (PS) and clearance related CD markers, including CD35, CD47, CD55 and CD59 on the surface of the RBCs, were detected by flow cytometry at 0h and 24h after drugs treatment. The proportion of acanthocytes by microscope also at 0h and 24h after drugs treatment was calculated. The phagocytosis of drug-treated RBC was detected by monocyte monolayer assay (MMA). Untreated RBCs were incubated in PBS by the same condition as a negative control.The influence of β-lactam antibiotics on RBC aging and clearance by all the results above was studied. 【Results】 Compare to the untreated RBCs, the drug treated RBCs showed a higher PS level on the cell surface. The results showed by percentage as following(0 h vs 24 h): Penicillin 9.42% vs 93.30%, Cefepime 3.88% vs 57.27%, Cefoperazone 4.71% vs 75.75% and Ceftazidime 3.05% vs 43.19%. The acanthocytes ratio was as following(0 h vs 24 h): Penicillin 7.33% vs 86%, Cefepime 2.67% vs 52.67%, Cefoperazone 3.33% vs 67.67% and Ceftazidime 3.33% vs 90.67%. On the opposite, the clearance related CD markers, showed an obviously lower level after drugs treated(0 h vs 24 h): CD35: Penicillin 7.36% vs 11.87%, Cefepime 0.14% vs 28.51%, Cefoperazone 11.85% vs 21.55% and Ceftazidime 7.63% vs 8.73%; CD47: Penicillin 1.22% vs 9.13%, Cefepime 1.80% vs 0.86%, Cefoperazone 0.08% vs 6.85% and Ceftazidime 1.54% vs 5.50%; CD55: Penicillin 14.46% vs 44.31%, Cefepime 17.27% vs 38.41%, Cefoperazone 19.28% vs 33.28% and Ceftazidime 14.62% vs 34.13%; CD59: Penicillin 4.71% vs 20.56%, Cefepime 4.03% vs 7.60%, Cefoperazone 5.91% vs 22.38% and Ceftazidime 5.93% vs 30.89%. Drug-treated RBCs attached more to monocytes than untreated RBCs. 【Conclusion】 The β-lactam antibiotics could induce the changing of PS and the clearance of related CD markers on surface of RBCs. They also could lead acanthocytes and make the RBCs more susceptible to phagocytosis by monocytes. The β-lactam antibiotics could promote the RBCs aging and clearance, which might deteriorate the DIIHA.

Preclinical characterization and comparison between CD3/CD19 bispecific and novel CD3/CD19/CD20 trispecific antibodies against B-cell acute lymphoblastic leukemia: targeted immunotherapy for acute lymphoblastic leukemia / 医学前沿

The CD19-targeting bispecific T-cell engager blinatumomab has shown remarkable efficacy in patients with relapsed/refractory B-cell precursor acute lymphoblastic leukemia. However, several studies showed that blinatumomab has a short plasma half-life due to its low molecular weight, and thus its clinical use is limited. Furthermore, multiple trials have shown that approximately 30% of blinatumomab-relapsed cases are characterized by CD19 negative leukemic cells. Here, we design and characterize two novel antibodies, A-319 and A-2019. Blinatumomab and A-319 are CD3/CD19 bispecific antibodies with different molecular sizes and structures, and A-2019 is a novel CD3/CD19/CD20 trispecific antibody with an additional anti-CD20 function. Our in vitro, ex vivo, and in vivo experiments demonstrated that A-319 and A-2019 are potent antitumor agents and capable of recruiting CD3 positive T cells, enhancing T-cell function, mediating B-cell depletion, and eventually inhibiting tumor growth in Raji xenograft models. The two molecules are complementary in terms of efficacy and specificity profile. The activity of A-319 demonstrated superior to that of A-2019, whereas A-2019 has an additional capability to target CD20 in cells missing CD19, suggesting its potential function against CD19 weak or negative CD20 positive leukemic cells.

Practice and analysis of international applications for scientific and technological innovation achievements in research hospitals: Take Zhongshan Hospital, Fudan University as an example / 中华医学科研管理杂志

Objective:With the fast increasing of patent applications and authorizations in the field of pharmaceutical industry in China, it is of great significance to deploy the international market in advance. This study takes the overseas patent application work of Zhongshan Hospital, Fudan University as an example, to explore feasible methods that suitable for research-oriented hospitals to help high-value patents enter the international market.Methods:Based on the working procedures and common ways of overseas patents declared by Zhongshan Hospital, Fudan University from 2018 to 2020, this study analyzes the process of international applications of scientific of technological innovation achievements of research-oriented hospitals and summarizes the elements of patent application through working practices.Results:A standardized international patent application approach in public hospitals should include the following six parts: value evaluation before international patent filing, selection of the country when entering the national phase, professional assistance of patent agency company, hospital internal audit process, multi-channel access to intellectual property management and operating capital support, professional talent team construction.Conclusions:A set of standardized procedures for the application of overseas patents in Zhongshan Hospital, Fudan University can help high-value patents enter the advanced market, which is operable and worthy for further generalization in other research hospitals.

-D- phenotype screening by polybrene method / 中国输血杂志

【Objective】 To develop a novel screening reagent for -D- phenotype preliminary screening based on the difference in RhD antigen expression level of -D- phenotype and normal RhD phenotype. 【Methods】 RhD antigen expression of -D-phenotype and Rh D-- gene carrier were detected by flow cytometry. By adjusting the concentration of polybrene in the screening system, the red blood cells with high RhD antigen expression level agglutinated, and the preliminary screening of the -D-phenotype and its gene carriers was realized. 【Results】 According to the quantitative results of immunofluorescence intensity (MFI) analysis by flow cytometry, the expression level of RhD antigen in -D- phenotype cells (284 360±16 698, n=3) was about 3 times normal RhD positive cells (98 642±35 908, n=9)(P<0.01), while RhD antigen expression level of RhD-- gene carrier (181 109±39 455, n=4) was about 2 times normal RhD positive cells(P<0.01). RhD antigen expression (144 538±227 445, n=7) of the positive cells screened by 15 μL 3% fresh red blood cell suspension and screening system 35 μL (1 μL IgG anti-D, 29 μL polybrene polybrene, and 5 μL low ionic strength solution) was about 1.5 times normal RhD positive cells. 【Conclusion】 The polybrene preliminary screening system, which can be used for high-throughput screening of -D- phenotype, is a reliable technical method for frequency study of this phenotype.

The role of miR-492 in the regulation of OK blood group antigen expression on red blood cells / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate whether miR-492 is involved in the post-transcriptional regulation of OK blood group antigen expression on red blood cells.</p><p><b>METHODS</b>Two 3'-UTR fragments of the BSG gene were synthesized with a chemical method, which respectively encompassed the BSG rs8259 TT or BSG rs8259 AA sites. The fragments were added with Xho I and Not I restriction enzyme cutting sites at both ends and cloned into a pUC57 vector, which in turn was constructed into a psiCHECK-2 vector and verified by sequencing. K562 cells were transfected with various combinations of miR-492 mimic and constructed psiCHECK2-BSG-T or psiCHECK2-BSG-A recombinant plasmid. A blank control group was set up. Each transfection experiment was repeated three times. The activity of Renilla reniformis luciferase was determined and normalized with that of firefly luciferase, and detected with a dual-luciferase reporter assay system. The data were subjected to statistical analysis.</p><p><b>RESULTS</b>The sequencing results confirmed that the recombinant psiCHECK2 plasmids containing the BSG rs8259 TT or rs8259 AA sites were constructed successfully. The results of dual-luciferase report gene detection showed that the miR-492 mimic could significantly inhibit psiCHECK2-BSG-T at a concentration over 100 nmol/L. However, it could not inhibit psiCHECK-BSG-A.</p><p><b>CONCLUSION</b>miR-492 may be involved in the regulation of OK antigen expression on red blood cells with the BSG rs8259 TT genotype.</p>

Current smoking prevalence and influencing factors among university students in Shanghai / 上海交通大学学报（医学版）

Objective · To understand current smoking status and analyze its influencing factors among university students in Shanghai, and provide reference and guidance for further efforts of tobacco control in campus. Methods · A total of 4816 students from 19 col eges in Shanghai were selected by stratified multi-stage cluster sampling and the sample size in each part was decided by proportion of col eges and types of specialities. Self-administered questionnaire was conducted to understand the current tobacco use and its influencing factors were analyzed. Results · The overal smoking prevalence of col ege students in Shanghai was 5.80%. General y, smoking prevalence of junior col ege students was higher than that of undergraduates (11.27% vs 3.68%, P<0.05) and smoking prevalence of male students was higher than that of females (11.10% vs 0.95%, P<0.05). Nonsmokers endorsed higher awareness on the harm of smoking and second-hand smoking than smokers(P<0.01). Besides, nonsmokers had a more positive attitude towards tobacco control policy than smokers (P<0.01). Students who studied in the junior college, males, in the senior grade, majored in liberal arts, with higher monthly living expenses, held negative attitude in raising cigarette prices and supporting of establishing smoke-free campuses were more likely to be smokers. Conclusion · Tobacco use among university students in Shanghai deserves attention. It is necessary to carry out systematic and in-depth education to prevent and reduce smoking among the university students.

Solid-phase screening of antibodies in blood and its effect on clinical blood transfusion / 中国输血杂志

Objective Detecting antibodies against RBCs,platelets,lymphocytes,and neutrophils by a solid-phase antibodies screening system.Methods mono-layer blood cell immobilized in the bottom of U-microplate respectively to prepare the solid-phase antibody screening system.Then detecting the antibodies exist or not in 2 150 random blood donor and 440transfusion patients' samples.Analyze the influence of antibodies against blood cells to the transfusion effect.Results 53RBC antibodies,22 platelet antibodies,13 lymphocyte antibodies,55 neutrophil antibodies were detected in random blood donor samples;31 RBC antibodies,38 platelet antibodies,24 lymphocyte antibodies,43 neutrophil antibodies and 115 mixed (two or more antibodies against RBC,platelets,lymphocytes or neutrophils exist at the same time) were detected in transfusion patients' samples after detection.233 suspected adverse reaction happened after transfusion,which antibodies were detected in 133 samples.Conclusion The antibodies against blood cells solid-phase screening system can applied in pre-and post-transfusion detection.The solid-phase screening method is more sensitivity than serologic tube test and flow cytometry.Existing of antibodies influence the transfusion effect.

Development of a multiplex polymerase chain reaction system for screening low-frequency blood group antigens K and Ytb / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>A multiplex PCR system for screening rare blood group antigens K and Yt(b) was constructed to study the distribution of the two blood groups in a Uygur population in Xinjiang, China.</p><p><b>METHODS</b>Sequence-specific primers (SSP) were designed based on single nucleotide polymorphism sites of KEL and ACHE alleles encoding the two blood group antigens. The system was designed for simultaneously detecting the two antigens by optimizing the PCR reaction. Three hundred and sixty-two randomly selected healthy individuals were screened. Products of PCR were further analyzed for heterozygosity.</p><p><b>RESULTS</b>The system was set up successfully. No KK sample was identified and 9 K+ k+ , 41 Yt (a+ b+ ), 4 Yt (a- b+ ) were found among the 362 samples.</p><p><b>CONCLUSION</b>The established PCR-SSP based multiple PCR system is efficient to screen the rare blood group antigens K and Yt(b). The information of rare blood donors obtained from the screening can be used to improve the capability of compatible transfusion.</p>

Screening of rare blood group Lu(a-b-) phenotype and study of its molecular basis in ethnic Han Chinese from Shanghai region / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To study the frequency of rare blood group Lu(a-b-) phenotype in a population from Shanghai region, and to explore the molecular basis of Lu(a-b-) by detecting the Lu and Lu relative mediator gene EKLF/KLF1.</p><p><b>METHODS</b>Donors from Shanghai region were screened for Lutheran blood group by monoclonal anti-Lub using serological methods. Individuals with Lu(b-) were determined Lua, P1 and i antigens. Fifteen exons of the LU gene and 3 exons of the EKLF/KLF1 gene for the identified Lu(a-b-) samples were amplified and sequenced.</p><p><b>RESULTS</b>Ten Lu(a-b-) donors were obtained from 44 331 donors from Shanghai region. No homozygous or heterozygous mutations were found in the LU gene, whilst 7 mutations in EKLF/KLF1 gene were identified in the 10 samples.</p><p><b>CONCLUSION</b>The frequency of rare Lu(a-b-) blood group in Shanghai was approximately 0.02%, and all the individuals had an In(Lu) phenotype. The molecular basis of such samples may be related to mutations in the EKLF/KLF1 gene.</p>

Application of multiplex PCR for the screening of genotyping system for the rare blood groups Fy(a-), s-,k-,Di(b-) and Js(b-) / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To screen rare blood groups Fy(a-), s-, k-, Di(b-) and Js(b-) in an ethnic Zhuang population.</p><p><b>METHODS</b>Sequence-specific primers were designed based on single nucleotide polymorphism (SNP) sites of blood group antigens Fy(b) and s. A specific multiplex PCR system I was established. Multiplex PCR system II was applied to detect alleles antigens Di(b), k, Js(b)1910 and Js(b) 2019 at the same time. The two systems was were used to screen for rare blood group antigens in 4490 randomly selected healthy donors of Guangxi Zhuang ethnic origin.</p><p><b>RESULTS</b>We successfully made the multiplex PCR system I. We detected the rare blood group antigens using the two PCR system. There are five Fy(a-), three s(-), two Di(b-) in 4490 Guangxi zhuang random samples. The multiplex PCR system I has achieved good accuracy and stability. With multiplex PCR systems I and II, 4490 samples were screened. Five Fy(a-), three s(-) and two Di(b-) samples were discovered.</p><p><b>CONCLUSION</b>Multiplex PCR is an effective methods, which can be used for high throughput screening of rare blood groups. The rare blood types of Guangxi Zhuang ethnic origin obtained through the screening can provide valuable information for compatible blood transfusion. Through screening we obtained precious rare blood type materials which can be used to improve the capability of compatible infusion and reduce the transfusion reactions.</p>

Improving the efficacy of blood transfusion by choosing an appropriate method for compatibility test / 中华检验医学杂志

Blood typing and compatibility testing directly affect curative effects of blood transfusion.To avoid severe blood transfusion reactions, more and more new immunohematology technologies and methods are becoming gradually applied to blood transfusion laboratories.Gel test, monocyte monolayer assay and analysis of blood group genes have been confirmed to be beneficial on improving the sensitivity and automatization.Some technologies can predict red cell survival in vivo more accurately.It can improve the efficacy of blood compatibility before transfusion by using these technologies.There is no general methods used in solving of complex serological problems, but medical staffs can provide appropriate blood for different sufferers by using different technologies at the right time.

An overview of the studies on combined electric and acoustic stimulation of auditory system / 生物医学工程学杂志

This paper discusses the technology of combined electric and acoustic stimulation (EAS) of the auditory system, which is a new therapy for the patients suffering from severe to profound high- and mid-frequency hearing loss but still having their low-frequency hearing. EAS uses hearing aid and cochlear implant technology together in the same ear. The hearing aid acoustically amplifies at low-frequencies, while the cochlear implant electrically stimulates at mid- and high-frequencies. The inner ear processes acoustic and electric stimuli simultaneously. This technique can provide substantial benefit in speech understanding for individuals with severe high-frequency hearing loss and can maintain their residual lower-frequency acoustic hearing. The study of EAS would significantly enhance the conventional cochlear implant therapy and benefit the patients afflicted with severe to profound hearing loss.

Advances in the study of virtual channels of cochlear implant / 生物医学工程学杂志

This paper discusses virtual channels of cochlear implant, which is produced by simultaneous or sequential activation of adjacent cochlear implant electrodes. Virtual channels create and transfer more available spectral pitch information with the limited number of fixed electrodes, which can be recognized as pitch percepts intermediate to those produced by each electrode separately. This technique not only utilizes the interaction of electrodes but also increases the number of place-pitch steps available to cochlear implant listeners. Virtual channels could be used to realize speech recognition in noisy environment, in enjoying music, and in understanding Chinese language. The study of virtual channels would significantly enhance the traditional cochlear implant therapy and benefit people suffering severe to profound hearing loss.

Different KEL gene mRNA transcripts in reticulocyte and non-reticulocyte cells / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the difference of the transcripts between reticulocyte and non-reticulocyte cells in human blood.</p><p><b>METHODS</b>Genomic DNA, reticulocyte RNA and total RNA of K-, K+ and Kell-null(K0) were extracted, then PCR, reverse transcription-PCR(RT-PCR) and nested PCR followed by sequencing or cloning-sequencing were used to analyze the KEL gene mRNA exons 1-19 and exons 2-8. Four kinds of monoclonal antibodies were labeled to detect the expression of Kell glycoprotein on red cells or leukocytes with flow cytometry.</p><p><b>RESULTS</b>In reticulocyte, only one normal KEL transcript faithful to the genomic structure was found in all tested samples except K0 which had 4 different transcripts. Sequence analysis of exons 2-8 of total RNA confirmed the alternative KEL transcripts existed in different samples, mostly caused by abnormal splicing, among them, skipping of exon 3 and a 16 bp insertion of intron 6 at the beginning of exon 7 were the most frequent. Although only one band was observed after amplifying the exons 1-19 from total RNA, the sequencing result showed it was a mixture of different sequences. There was strong expression of Kell glycoprotein on red cells except K0, but no or low expression on leucocytes by flow cytometry.</p><p><b>CONCLUSION</b>Alternative transcripts of KEL gene exist in different cells, which would be responsible for different Kell glycoprotein expression patterns on different cells. This study suggested that reticulocyte RNA was more suitable than total RNA for molecular study of KEL gene transcription.</p>

Genotyping of human platelet antigen system 5 by PCR restriction fragment length polymorphism / 中华检验医学杂志

Objective To develop a polymerase chain reaction restriction fragment length polymorphism (PCR RFLP) method using designed primers for determining the genotype of humen platelet antigens (HPA)5 system. Methods HPA 5 system of 25 healthy blood donors were genotyped using PCR RFLP method. The results obtained by PCR RFLP were compared with those determined by allele specific oligonucleotid hybridization (PCR ASO). Results The results of HPA 5 system obtained by PCR RFLP in 25 health donors were as follows: 24 of aa, 1 of ab and 0 of bb. All were in good agreement with those determined by PCR ASO. Conclusions Because PCR RFLP method is plain, fast and reliable for HPA 5 system genotyping, it is suitable for the diagnosis and therapy of neonatal alloimmune thrombocytopenia, posttransfusion purpura, platelet transfusion refractoriness and so on..

Molecular study on Ax subgroup in Chinese population / 中国输血杂志

Objective To study Ax subgroup’s molecular characteristics in Chinese Han population. Methods Eight samples suspected as Ax subgroup were analyzed and duplex PCR RFLP test was used to determine the primary ABO genotypes. These samples were then analyzed by another PCR RFLP test to identify whether there was an nt646 “T” to “A” mutation within the exon 7 of ABO gene, which was a known mutation related to most Ax phenotypes. Samples with discrepancy between serological and gene typing were chosen for further T A cloning and sequence analysis. Results Four out of all tested samples had the known nt646 “T” to “A” mutation. An A *weak01 allele including nt407 and nt467 “C” to “T” mis sense mutation was detected in this study. Moreover, a novel Ax allele with a new single nucleotide C to T mutation was detected at nt745. Another 2 unrelated samples were suspected as AxB through serological test, both of which contained higher quantities of anti A and showed strong agglutination with anti H. And their initial genotypes were BO, and sequence analysis clarified that both had normal O gene and novel nt640 “A” to “G” mutation in their B alleles. Conclusion The novel Ax alleles, one kind of novel B(A) allele and one A *weak01 allele in Chinese Han individuals,have been detected. B(A) phenotypes should have their molecular biology bases as well as other ABO subgroups.