[Retrospective clinical study on cryopreservation-free integrated autologous hematopoietic stem cell transplantation model for newly diagnosed multiple myeloma].

Objective: To explore the efficacy and safety of cryopreservation-free integrated autologous hematopoietic stem cell transplantation (HSCT) model for patients with multiple myeloma. Methods: A total of 96 patients with newly diagnosed multiple myeloma (NDMM) between July 31, 2020, and December 31, 2022, were retrospectively analyzed, of which 41 patients in the observation group received integrated non-cryopreserved transplantation mode. After hematopoietic stem cells were mobilized and collected, melphalan was started immediately for pre-transplant conditioning, and non-cryopreserved grafts from the medical blood transfusion refrigerator were directly injected intravenously into the patient within 24-48 h after the melphalan conditioning. The control group consisted of 55 patients who received traditional transplantation mode. After hematopoietic stem cells were collected, stem cell cryopreservation was performed in liquid nitrogen, and then the transplant plans were started at the right time. All patients received mobilization of autologous hematopoietic stem cells using the G-CSF combined with the plerixafor. Results: ① A total of 34 patients (82.9% ) with VGPR plus CR in the observation group were significantly higher than 33 patients (60.0% ) in the control group (P=0.016). ②Compared with the control group, the incidence of grade 1 oral mucosal inflammation was higher in the observation group (P<0.001) ; however, the incidence of grades 2 and 3 oral mucosal inflammation was lower (P=0.004, P=0.048), and neither group experienced grade 4 or above oral mucosal inflammation. The incidence of grade 1 diarrhea was higher in the observation group (P=0.002), whereas the incidence of grade 3 diarrhea was lower (P=0.007). No statistically significant difference was observed in the incidence of grade 4 diarrhea (P=0.506), and neither group experienced grade 5 diarrhea. ③ The incidence of bacterial infection in the observation group was lower than that in the control group (34.1% vs 65.5%, P=0.002), whereas no statistically significant difference was observed in the incidence of fungal infection (29.3% vs 31.4%, P=0.863) and viral infection (4.88% vs 3.64%, P=0.831). ④No statistically significant difference was observed in the implantation time of granulocytes and platelets between the observation and control groups [10 (8-20) days vs 11 (8-17) days, P=0.501; 13 (10-21) days vs 15 (10-20) days, P=0.245]. ⑤ All patients did not receive lenalidomide treatment 100 days post-transplantation. At 30 days post-transplantation, the CTL, NK, and Th cell counts in the observation group were lower than those in the control group (P<0.001, P=0.002, P=0.049), and the NKT cell counts were higher than those in the control group (P=0.024). At 100 days post-transplantation, the CTL, NKT, and Th cell counts in the observation group were higher than those in the control group (P=0.025, P=0.011, P=0.007), and no statistically significant difference in NK cell counts was observed between the two groups (P=0.396). ⑥ The median follow-up was 18 (4-33) months. The overall 2-year survival rates of the observation and control groups post-transplantation were 91.5% and 78.2%, respectively (P=0.337). The recurrence-free survival rates were 85.3% and 77.6%, respectively (P=0.386), and the cumulative recurrence rates were 9.8% and 16.9%, respectively (P=0.373) . Conclusion: In NDMM, the cryopreservation-free integrated autologous HSCT model can achieve similar therapeutic effects as traditional transplantation models, with lower rates of severe mucosal inflammation and infection compared with traditional transplantation models.

[Effect of REG3A on proliferation and invasion of glioma cells by regulating PI3K/Akt signaling pathway].

Objective: To investigate the effects of regenerating islet-derived protein 3A (REG3A) on the proliferation and invasion of glioma cells and its molecular mechanism. Methods: Five low-grade, five high-grade glioma tissues and ten adjacent tissues from glioma patients who underwent surgery at Linyi People's Hospital from October 17, 2017 to October 18, 2018 were collected. Human glioma cell lines (SF295, U251, TG905, A172, CRT) and a primary glioma cell line PT-1 were cultured in vitro. The protein and mRNA expressions of REG3A in these tissues and glioma cell lines were detected by Western blot and reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR). SF295 cells were infected with lentivirus and labeled as REG3A plasmid transfection group, and the TG905 cells were transfected with si-REG3A by liposome transfection reagent and labeled as si-REG3A transfection group. At the same time, the empty transfection control and blank control groups were set up. Glioma cells were treated with REG3A recombinant protein alone or in combination with Akt1/2 inhibitors. Cell counting kit-8 (CCK-8) and cell scratch assay were used to detect cell proliferation and invasion, respectively. Western blot was used to detect the protein expression of N-cadherin, vimentin and phosphorylation of Akt (p-Akt) in REG3A overexpressed and knockdown glioma cells. Results: RT-qPCR results showed that the mRNA expression levels of REG3A in glioma cells in each group were U251 (2.129±0.13), TG905 (2.22±0.59), CRT (5.02±0.31), A172 (6.62±1.34) and PT-1 (9.18±0.61), respectively, higher than its expression in SF295 cells (1.00±0.18, P<0.001). The mRNA expression level of REG3A in high-grade glioma tissue samples (3.18±2.92) was higher than that in the control group (1.00±1.14, P=0.031) and low-grade glioma group (0.90±0.67, P=0.014). The results of western blot and immunohistochemical staining were consistent with that of RT-qPCR. The migration rate of cells in si-REG3A transfection group [(60.57±5.30)%] was lower than that of the empty transfection group [(84.18±13.63)% (P=0.038)] and blank control group [(79.65±12.09)% (P=0.076)]. The results of the scratch experiment showed that the migration rate of cells in REG3A plasmid transfected cells in the SF295 group was (96.05±6.41)%, which was significantly higher than that of empty transfected cells [(74.47±8.23)%, P=0.021)]. REG3A recombinant protein could up-regulate the expression of N-cadherin, vimentin and p-Akt in SF295 cells. Compared with the control group [(100.00±2.53)%], the proliferation rate in the REG3A recombinant protein group [(117.70±10.24)%] was significantly up-regulated, and the proliferation rate in the REG3A recombinant protein+ Akt inhibitor group [(98.31±3.64)%] was significantly lower than that of the REG3A recombinant protein group (P=0.017). The migration rate of the REG3A recombinant protein+ Akt inhibitor group was (63.35±4.06)%, which was significantly lower than (89.26±11.07)% of the REG3A recombinant protein group (P=0.019). Conclusion: REG3A can promote the proliferation and invasion of human glioma cells by activating the PI3K/Akt signaling pathway.

[Clinical analysis of 226 cases of deviated nose with deviated nasal septum treated by endoscopic assisted functional rhinoplasty].

Objective: To explore the method and effect of endoscopic assisted functional rhinoplasty for patients with deviated nose and deviated nasal septum, which achieve correction of nasal morphology and ventilation dysfunction. Methods: The clinical data of 226 patients with deviated nose and deviated nasal septum from June 2009 to February 2022 who were treated by endoscopic assisted functional rhinoplasty in the Affiliated Hospital of Qingdao University were analyzed retrospectively. There were 174 males and 52 females, with the age ranging from 7 to 67 years old. The effect was evaluated by subjective and objective evaluation methods. SPSS 27.0 software was used for statistical analysis. Results: All patients were followed up for 6 to 24 months, 174 cases were cured (174/226, 76.99%), 52 cases were effective (52/226, 23.01%), and the total effective rate was 100% (226/226). The difference between preoperative and postoperative facial appearance deviation was statistically significant ((6.84±2.25)mm vs (1.82±1.05)mm, t=38.94, P<0.001), and the nasal ventilation function of all patients was improved. Conclusions: Endoscopic assisted functional rhinoplasty for the patients with deviated nose combined with deviated nasal septum has the advantages of clear surgical field, fewer complications, and good result. It can achieve the purpose of simultaneous correction of nasal and ventilation dysfunction, which is recommended for popularizing in clinical application.

[Genotype-phenotype analysis of a homozygous familial hypercholesterolemia pedigree].

Objective: To analyze the genetic characteristics of a five generations pedigree with homozygous familial hypercholesterolemia (HoFH). Methods: Prospective study. Twenty family members included a proband diagnosed as familial hyperlipidemia at the cardiology Department of Xi'an Children's Hospital in October 2018 were research object. Clinical data were collected. Genome DNAs were extracted. Whole exons sequencing was performed on the proband using target capture next generation sequencing. Candidate gene mutation sites identified by bioinformatics were verified by Sanger sequencing in the family members. The genotype-phenotype correlation of the pedigree was analyzed between heterozygous mutation carriers and non-carriers. Results: The proband was a 7-years and 10-month-old boy. He was born with a roundgreen bean size yellow skin protuberance in the skin of the coccyx. Since the age of 3-4 years old, xanthoma-like lesions with a diameter of 0.5-1.5 cm gradually appeared in the skin of bilateral elbow joints, knee joints and Achilles tendon. The height, weight and intellectual development of the child were the same as those of normal children at the same age. No similar xanthoma-like lesion was found in the other family members. The proband's total cholesterol (TC) reached 18.16-21.24 mmol/L, and his low density lipoproteincholesterol (LDL-C) was 14.08-15.51 mmol/L. Carotid ultrasonography showed diffuse sclerotic plaques in bilateral carotid and vertebral arteries, and color Doppler echocardiography revealed aortic valve thickening and calcification. Gene testing identified that the proband carried a homozygous mutation C. 418G>A (p. E140K) in LDLR gene inherited from his parents who had a consanguineous marriage and carried a heterozygous mutation of LDLR-E140K, respectively.The TC, LDL-C and apolipoproteinB (ApoB) of LDLR-E140K gene heterozygous carriers ((8.40±0.13), (6.79±0.01) and (1.95±0.05) mmol/L, respectively) were significantly higher than those of non-carriers ((4.59±0.28), (3.35±0.39) and (0.86±0.10) mmol/L, t=7.269, 4.595, 6.311, respectively, P<0.05). Conclusions: LDLR-E140K gene homozygous mutation is first reported to be associated with most severe phenotype HoFH. The genotype-phenotype analysis of the pedigree shows that the clinical phenotype of the proband with homozygous mutation is the most serious, and all the heterozygous mutation carriers present with hypercholesterolemia phenotype. The investigation confirms that LDLR-E140K is the pathogenic variation of familial hyperlipidemia.

Roles of the Nrf2/HO-1 pathway in the anti-oxidative stress response to ischemia-reperfusion brain injury in rats.

OBJECTIVE: The aim of this study was to investigate the roles of the Nrf2/HO-1 pathway in the responses to the oxidative stress created by ischemia-reperfusion brain injury in rats. MATERIALS AND METHODS: 54 healthy, adult, male SD rats were included in the study. Eighteen (18) rats were placed in the sham group. The ischemia-reperfusion model was created in the other 36 rats, among which 18 received injections of Nrf2 agonist before the surgery. The suture method was used to create artery occlusions in the right brain of the rats; and reperfusion was done after 90-minute ischemia (MCAO); while no suture was inserted in the sham group. At 3, 6, 12, 24, 48 and 72 hours after the modeling, their neurological functions were evaluated. Also, at different time points, rats were decapitated, and their fresh brain tissues were used to detect the infarct volume percentages by TTC staining and the brain water contents by the dry-wet weight method. The SOD contents in the brain tissue were measured by Xanthine oxidase assay. RT-PCR was used to detect the mRNA expression of HO-1 in the brain tissues, and western blot method was used to detect the expression level of HO-1 and Nrf-2. RESULTS: The rats in the sham group had no obvious neurological defects; while those in the MCAO group showed significant neurological defects at all time points. The MCAO group had higher neurological evaluation scores than the sham group. TTC staining showed that infarct in the MCAO group kept increasing over time and peaked at 24h. Measurements of SOD found that the sham group had the highest SOD among the three groups, and showed no significant fluctuation over time. The MCAO group had much lower SOD activities than the sham group at all the time points. The higher the level of HO-1mRNA and protein expression in the brain tissue of rats in each group, the higher the degree of brain injury, but the lower the level of Nrf2 protein expression and the lower degree of brain injury. Nrf2 agonist markedly improved all these indicators in the rats which underwent the MCAO surgery. CONCLUSIONS: The expression of HO-1 after ischemia-reperfusion brain injury may contribute to the increased infarct volume. Activation of Nrf2 could improve the prognosis of ischemia-reperfusion brain injury.

Metabolic syndrome is an independent prognostic factor for endometrial adenocarcinoma.

OBJECTIVE: To study the association between metabolic syndrome (MS) and the prognosis of patients with endometrial adenocarcinoma. METHODS: A total of 385 patients with endometrial adenocarcinoma in the Department of Gynecologic Oncology, at the Zhejiang Cancer Hospital in China, between January 2001 and December 2008 were chosen. The deadline for the completion of follow-up was December 2013. The overall survival (OS) of the patients with MS was analyzed by the Kaplan-Meier method. Various clinical characteristics (e.g., clinical and surgical stage, vascular invasion, histological grade, tumor size, age at start of the first treatment, and lymphatic metastasis) related to the prognosis of endometrial adenocarcinoma were also evaluated. RESULTS: A univariate analysis demonstrated that the OS rate of the patients with endometrial adenocarcinoma with MS was significantly worse than that of the patients without MS for all 385 patients (P = 0.001). Multivariate Cox proportional hazards regression analyses showed that stage (P = 0.001), lymphatic metastasis (P = 0.021), and MS (P = 0.049) were independent prognostic factors for endometrial adenocarcinoma. Furthermore, statistical analyses demonstrated that MS was closely related to stage (P = 0.021), grade (P = 0.022), vascular invasion (P = 0.044), tumor size (P = 0.035), and lymphatic metastasis (P = 0.014) but not with age at start of the first treatment (P = 0.188). Finally, according to the univariate analysis of the OS rate of 129 cases of endometrial adenocarcinoma with MS, stage (P = 0.001), vascular invasion (P = 0.049), tumor size >2 cm (P = 0.028), lymphatic metastasis (P = 0.002), and CA19-9 value >37 U/m (P = 0.002) all showed significantly low P values for OS. CONCLUSION: Metabolic syndrome is an independent prognostic factor for endometrial adenocarcinoma.

Surgical management and clinical outcome of gastrointestinal stromal tumor of the colon and rectum.

BACKGROUND: A standardized treatment for gastrointestinal stromal tumors (GIST) of the colon and rectum has not been clearly established. The objective of this study is to examine our experience in patients with colorectal GISTs and review the appropriate surgical management. METHODS: The medical records of patients with colorectal GIST treated in our institution between 1995 and 2005 were reviewed. The malignant potential of the GIST was assessed with the current consensus criteria defined by the National Institutes of Health. Clinical parameters were also evaluated to determine prognostic factors. RESULTS: There were 10 male and 7 female patients, with a median age of 64 years (range: 19 - 84). Bloody stool and abdominal pain were the most commonly reported symptoms in colorectal GISTs. There were 7 colonic GISTs and 10 rectal GISTs. Sixteen patients underwent surgery with a margin of negative resection including 12 radical surgical resections, 3 transanal wide excisions, and one colonoscopic excision. Pathological results revealed a high risk in 8 patients (47.1 %), an intermediate risk in 4 (23.5 %), a low risk in 3 (17.6 %), and a very low risk in 2 (11.8 %). Three patients (3 / 16, 18.6 %) developed disease relapse after primary radical resection. All the three patients were high-risk rectal GISTs, accounting for 42.9 % (3 / 7) in the high-risk group. The median time to disease relapse was 15.7 months (range: 6 - 24). Cox regression analysis showed that variables including age, gender, and tumor size were not presenting statistically significant differences between groups of relapse and non-relapse patients. CONCLUSION: Non-high-risk colorectal GISTs bear a good prognosis after margin-negative surgery. Transanal wide excision for non-high-risk GISTs is mandatory if a complete resection can be performed. Abdominoperineal resection would be preserved only in patients with high risk or large non-high-risk lower rectal GISTs. The high-risk group has high incidence of relapse even though a complete resection was achieved. Adjuvant therapy with a tyrosine kinase inhibitor would be beneficial to these patients.

Quantitative peptidomics of pituitary glands from mice deficient in copper transport.

We previously described a method of quantitating levels of peptides in Cpe(fat)/Cpe(fat) mice using affinity chromatography to isolate peptide-processing intermediates and differential isotopic labeling/mass spectrometry. In the present study, we compared two different isotopic labels, acetic anhydride and succinic anhydride for detection and quantitation of peptides in wild type mice. As previously found for acetic anhydride, succinic anhydride efficiently labels all primary amines in various peptides. Of these two reagents, succinic anhydride provides better resolution between the heavy and light peaks of the labelled peptides due to a greater mass difference between the deuterated (heavy) and non-deuterated (light) form of this label (4 Da for succinate, 3 Da for acetate). Using succinic anhydride labeling, the accuracy of measuring 1:1 and 1:2 ratios of peptides in pituitary extracts was within 5% of the theoretical value for most peptides. The accuracy with succinic anhydride is comparable to the accuracy of acetic anhydride and more peptides could be detected and quantitated with succinic anhydride. The two labels were then used to examine pituitary peptides in mice with a defect in copper transport (Atp7a mice) vs wild type mice. Using succinic anhydride, 13 peptides could be detected, 12 of which matched the theoretical mass of known pituitary peptides. Five of the six peptides which contain C-terminal amide groups were significantly decreased in the Atp7a mice relative to wild type mice, whereas only one non-amidated peptide was significantly decreased in Atp7a mice. With acetic anhydride, only five peptides could be quantitated. The three peptides which contain C-terminal amide groups were decreased approximately 30% in the Atp7a mice. The selective decrease in amidated peptides in Atp7a mice is consistent with the copper-requirement of the enzyme that forms C-terminal amides.

Direct ligand-receptor complex interaction controls Brassica self-incompatibility.

Many higher plants have evolved self-incompatibility mechanisms to prevent self-fertilization. In Brassica self-incompatibility, recognition between pollen and the stigma is controlled by the S locus, which contains three highly polymorphic genes: S-receptor kinase (SRK), S-locus protein 11 (SP11) (also called S-locus cysteine-rich protein; SCR) and S-locus glycoprotein (SLG). SRK encodes a membrane-spanning serine/threonine kinase that determines the S-haplotype specificity of the stigma, and SP11 encodes a small cysteine-rich protein that determines the S-haplotype specificity of pollen. SP11 is localized in the pollen coat. It is thought that, during self-pollination, SP11 is secreted from the pollen coat and interacts with its cognate SRK in the papilla cell of the stigma to elicit the self-incompatibility response. SLG is a secreted stigma protein that is highly homologous to the SRK extracellular domain. Although it is not required for S-haplotype specificity of the stigma, SLG enhances the self-incompatibility response; however, how this is accomplished remains controversial. Here we show that a single form of SP11 of the S8 haplotype (S8-SP11) stabilized with four intramolecular disulphide bonds specifically binds the stigma membrane of the S8 haplotype to induce autophosphorylation of SRK8, and that SRK8 and SLG8 together form a high-affinity receptor complex for S8-SP11 on the stigma membrane.

Dual targeting of spinach protoporphyrinogen oxidase II to mitochondria and chloroplasts by alternative use of two in-frame initiation codons.

Protoporphyrinogen oxidase (Protox) is the final enzyme in the common pathway of chlorophyll and heme biosynthesis. Two Protox isoenzymes have been described in tobacco, a plastidic and a mitochondrial form. We isolated and sequenced spinach Protox cDNA, which encodes a homolog of tobacco mitochondrial Protox (Protox II). Alignment of the deduced amino acid sequence between Protox II and other tobacco mitochondrial Protox homologs revealed a 26-amino acid N-terminal extension unique to the spinach enzyme. Immunoblot analysis of spinach leaf extract detected two proteins with apparent molecular masses of 57 and 55 kDa in chloroplasts and mitochondria, respectively. In vitro translation experiments indicated that two translation products (59 and 55 kDa) are produced from Protox II mRNA, using two in-frame initiation codons. Transport experiments using green fluorescent protein-fused Protox II suggested that the larger and smaller translation products (Protox IIL and IIS) target exclusively to chloroplasts and mitochondria, respectively.

Molecular characterization and subcellular localization of protoporphyrinogen oxidase in spinach chloroplasts.

Protoporphyrinogen oxidase (Protox) is the last common enzyme in the biosynthesis of chlorophylls and heme. In plants, there are two isoenzymes of Protox, one located in plastids and other in the mitochondria. We cloned the cDNA of spinach (Spinacia oleracea) plastidal Protox and purified plastidal Protox protein from spinach chloroplasts. Sequence analysis of the cDNA indicated that the plastid Protox of spinach is composed of 562 amino acids containing the glycine-rich motif GxGxxG previously proposed to be a dinucleotide binding site of many flavin-containing proteins. The cDNA of plastidal Protox complemented a Protox mutation in Escherichia coli. N-terminal sequence analysis of the purified enzyme revealed that the plastidal Protox precursor is processed at the N-terminal site of serine-49. The predicted transit peptide (methionine-1 to cysteine-48) was sufficient for the transport of precursors into the plastid because green fluorescent protein fused with the predicted transit peptide was transported to the chloroplast. Immunocytochemical analysis using electron microscopy showed that plastidal Protox is preferentially associated with the stromal side of the thylakoid membrane, and a small portion of the enzyme is located on the stromal side of the chloroplast inner envelope membrane.

[Effects of adenovirus-mediated p16 and p53 genes transfer on apoptosis and cell cycle of lung carcinoma cells].

OBJECTIVE: To explore the synergistic inhibition effect and apoptosis induction of p16 and p53 genes on lung carcinoma cells. METHODS: E1-deficient and replication-defective recombinant p16 and p53 adenoviruses were generated by liposome-mediated co-transfection of recombinant plasmid pAdCMV-p16 or pAdCMV-p53 along with pJM17 and homologous recombination in 293 packaging cell. The lung cancer cell line H358, which had a homozygous deletion of p53 gene and no expression of p16 mRNA and protein, was infected with recombinant p16 and p53 adenovirus either individually or together. RESULTS: Immunohistochemical analysis showed that recombinant adenovirus could transfer p53 gene into tumor cell with 98% efficiency. Western blot indicated that p16 and p53 proteins were expressed at a high level in infected H358 cell. Inhibition effect of p53 gene on proliferation of H358 cell was weaker than that of p16 gene, and the combined use of both genes could completely prevent the proliferation of H358 cell. In situ end-labeling and flow cytometry indicated that p16 could result in G(1) arrest of cell cycle and did not induce H358 cells to undergo apoptosis; p53 also induced apoptosis of few cells besides G(1) arrest; and the simultaneous use of p16 and p53 genes could induce marked apoptosis of H358 cells. CONCLUSION: p16 and p53 genes possess the synergistic inhibiting effect on growth of lung cancer cells and can cooperate to induce apoptosis of H358 cell. The combined application of recombinant p16 and p53 adenoviruses can be used as a new strategy for cancer gene therapy.

Relationship between polyploidy and pollen self-incompatibility phenotype in Petunia hybrida Vilm.

Self-incompatibility in Solanaceae is controlled by a single multiallelic locus, the S-locus. The S-allele associated ribonucleases (S-RNases) in the pistil are involved in pollen rejection. In this work, we analyzed two newly isolated lines of Petunia hybrida, termed PB and PF. They both had the same set of S-RNases (SB1- and SB2-RNases), however the PB was a self-incompatible diploid while PF was a self-compatible tetraploid. Cross pollination tests between PB and PF indicated diploid pollen from PF lost the incompatibility phenotype. In order to clarify the effects of polyploidy on pollen phenotypic change, we artificially induced tetraploid plants from a diploid SB1SB2 heterozygote (= PB) and a diploid SB1SB1 homozygote. The obtained SB1 SB1SB1SB1 homoallelic tetraploid remained self-incompatible, whereas the SB1SB1SB2SB2 heteroallelic tetraploid became self-compatible. These data suggested that the diploid heteroallelic pollen lost the incompatibility phenotype and had the characteristics of self-compatibility with SB1SB2 style.

[Clinical significance of quantitative studies of cell nucleus in lymphocytes of the cerebrospinal fluid in leukemia].

The nuclear content, area and perimeter of the nucleus of lymphocytes in the C.S.F. were determined quantitatively by means of image analysis technique. 26 cases of central nervous system lymphocytic leukemia (CNLL), and 8 suspected cases were studied, other 56 cases who did not have leukemic and neoplastic diseases and had normal C.S.F. lymphocytes were taken as a control. Our data showed that all the mean nuclear content (MNC), mean nuclear area (MNA), mean nuclear perimeter (MNP), and the maximum and minimum nuclear contents of the 2 groups of former patients were obviously higher than those of the contral (P less than 0.01). These results presented suggestion that the image analysis technique can be used for differentiating the leukemic lymphocyte from normal one especially in suspected cases, and thus the diagnosis of CNLL might be improved.