The roles of branched-chain amino acids metabolism in tumorigenesis and progression.

Branched-chain amino acids (BCAAs), including leucine, valine, and isoleucine, play crucial roles in regulating metabolic balance and maintaining physiological functions in the body. Extensive studies have been focused on their implications in obesity, diabetes, and cardiovascular diseases. Nevertheless, accumulating evidence suggests that BCAAs metabolism also plays significant roles in tumorigenesis and progression. In this review, we overview recent progress of the study on BCAAs metabolism including its relationship with epigenetic regulation. Particularly, we discuss the metabolic reprogramming and metabolic sensing of BCAAs and its intermediate metabolites in tumor cells and microenvironment to decipher their functions. An enhanced understanding of the roles and mechanism of BCAAs metabolism in tumorigenesis and progression will contribute to development of novel therapeutic strategies against tumor.

Optimal dilation duration of 10 mm diameter balloons after limited endoscopic sphincterotomy for common bile duct stones: a randomized controlled trial.

Limited endoscopic sphincterotomy (EST) combined with endoscopic papillary balloon dilation (EPBD) is widely used. However, the optimal duration of small balloon dilation in choledocholithiasis remains controversial. We aimed to determine the optimal duration for 10 mm diameter balloon dilation after limited EST in choledocholithiasis. In this randomized controlled clinical trial, 320 patients were randomly assigned to receive small balloon dilation (10 mm in diameter) for 1 min (n = 160) or 3 min (n = 160) after deep bile duct cannulation. No significant difference in success rate of stone extraction between the two groups was observed. The incidence of post-ERCP pancreatitis (PEP) was higher in the 1 min group (10.6%) than in the 3 min group (4.4%) (P = 0.034). The logistic regression analysis showed that guidewire into the pancreatic duct, cannulation time > 5 min and 1 min balloon dilation were independent risk factors for PEP. There were no significant differences in other post-ERCP adverse events such as acute cholangitis, bleeding, perforation, etc. between the two groups. In conclusion, 3 min in duration was determined to be the optimal dilation condition for the removal of common bile duct stones.

Two undescribed steroids from Munronia pinnata (Wall.) W. Theob.

Two undescribed steroids, named (15 R)-2,15-dihydroxypregna-1,4-dien-3,16-dione (1) and 2,15-dihydroxypregna-1,4,14-trien-3,16-dione (2), were isolated from the aerial parts of Munronia pinnata (Wall.) W. Theob. The structure elucidation of two compounds was performed by using spectroscopic methods and comparing the literature. Compound 2 exhibited inhibitory effect against PTP-1B with an IC50 value of 152.07 ± 3.33 µM, and compound 1 was inactive.

Numerical simulation research of the transportation and distribution characteristics on sea surface of the microplastic released continuously for 12 years from China's coastal cities.

Based on the Lagrangian random walk particle tracking method and the global ocean reanalysis data, this study simulated the drift-diffusion process in ocean of microplastic particles (density less than seawater) discharged by coastal cities in China for 12 consecutive years. The results reveal that most of the microplastics (80.33%) essentially end up ashore or in the marginal seas around China, a small portion of microplastics (18.22%) enter the Sea of Japan and the Northwest Pacific Ocean via the Tsushima Strait and the Osumi-Kaikyo with the Kuroshio Tide, a very small portion of microplastics (1.45%) enter into the waters of Southeast Asian countries along with the west boundary current of South China Sea. The concentration distribution characteristics have obvious seasonal variation in the high concentration areas (the marginal seas around China and Sea of Japan). The mainly destination area of microplastics released in different cities is different.

Mechanisms of conservation tillage on nitrogen-fertilizer reduction and maize grain improvement in Mollisols of Northeast China: Insights from a 15N tracing study.

Conservation tillage is an important management practice to guarantee soil fertility in degraded Mollisols. It is still unclear, however, whether the improvement and stability of crop yield under conservation tillage can be sustainable with increasing soil fertility and reducing fertilizer-N application. Based on a long-term tillage experiment initiated in Lishu Conservation Tillage Research and Development Station by Chinese Academy of Sciences, we conducted a 15N tracing field micro-plot experiment to investigate the effects of reducing nitrogen application on maize yield and fertilizer-N transformation under long-term conservation tillage agroecosystem. There were four treatments, including conventional ridge tillage (RT), no-tillage with 0% (NT0), 100% (NTS) maize straw mul-ching, and 20% reduced fertilizer-N plus 100% maize stover mulching (RNTS). The results showed that after a complete cultivation round, the average percentages of fertilizer N recovery in soil residues, crop usage, and gaseous loss were 34%, 50%, and 16%, respectively. Compared with conventional ridge tillage, no-tillage with maize straw mulching (NTS and RNTS) significantly increased the use efficiency of fertilizer N in current season by 10% to 14%. From the perspective of N sourcing analysis, the average percentage of fertilizer N absorbed by crop parts (including seeds, straws, roots, and cobs) to the total N uptake reached nearly 40%, indicating that soil N pool was the main source of N for crop uptakes. In comparison with conventional ridge tillage, conservation tillage significantly increased total N storage in 0-40 cm by reducing soil disturbance and increasing organic inputs, and thus ensured the expansion and efficiency increment of soil N pool in degraded Mollisols. Compared with conventional ridge tillage, NTS and RNTS treatments significantly increased the maize yield from 2016 to 2018. In all, by improving fertilizer nitrogen utilization efficiency and maintaining the continuous supply of soil nitrogen, long-term management of no-tillage with maize straw mulching could achieve a stable and increasing maize yield in three consecutive growing seasons and simultaneously reduce environmental risks derived by fertilizer-N losses, even under the condition of 20% reduction of fertilizer-N application, and thus actualize the sustainable development of agriculture in Mollisols of Northeast China.

Feasibility and Safety of Transgastric Natural Orifice Transluminal Endoscopic Surgery in the Diagnosis of Ascites of Unknown Origin.

Objective: The purpose of this study was to evaluate the feasibility and safety of transgastric natural orifice transluminal endoscopic surgery (TG-NOTES) combined with biopsy in the diagnosis of unknown ascites. Method: This retrospective study used data from the first affiliated hospital of Nanchang university on 51 patients who were diagnosed with ascites of unknown origin between January 2013 and May 2019 and experienced peritoneal biopsy through TG-NOTES. The outcome measures included diagnostic accuracy and procedure-related adverse events. Results: TG-NOTES was performed successfully in 46 of 51 patients, tuberculous ascites in 38 cases, carcinomatous ascites in 4 cases, cirrhotic ascites in 1 case, and 3 cases showed no obvious abnormalities in pathological result. Five cases failed to be diagnosed because of abdominal adhesions. The diagnostic rate of TG-NOTES was 84.3%. There were no severe procedure-related adverse events and no mortality. All patients had good wound healing and no complaint of discomfort on follow-up. Conclusion: The majority of ascites of unknown origin can be expounded through TG-NOTES combined with biopsy without severe complication, therefore, it is a feasible and safe method to detect the cause of unexplained ascites.

Clinical analysis of 45 cases of perforation were identified during endoscopic retrograde cholangiopancreatography procedure.

Background: Endoscopic retrograde cholangiopancreatography (ERCP) has become an important method to diagnose and treat biliary-pancreatic diseases. Perforations are infrequent but serious complications can occur during ERCPs. However, it is unclear which patients are suitable for surgery and when these patients should receive surgery. Aim: To analyze the outcome of 45 patients with endoscopic retrograde cholangiopancreatography (ERCP) related perforation. Materials and methods: We retrospectively reviewed all 45 patients with ERCP-related perforation between January 2003 and December 2017, and observed the location and causes of perforation, treatment strategies, and mortality. Results: Twenty thousand four hundred and seventy-nine patients received ERCP procedures from January 2003 to December 2017 in our digestive endoscopy center. Forty-five patients suffered from ERCP-related perforations. The incidence rate of ERCP-related perforations was 0.22%. Twenty-six patients suffered from periampullary perforations, 15 patients suffered from duodenal wall perforations, 1 patient suffered from a fundus perforation, 1 patient suffered from a residual gallbladder duct perforation, 1 patient suffered from a papillary diverticulum perforation, and 1 patient suffered from an intrahepatic bile duct perforation. Six patients with duodenal perforations underwent surgery, and the other patients received conservative treatment. One patient with a duodenal perforation and ERCP-related pancreatitis died of heart failure, and all the other patients recovered. The mortality rate was 2.2%. Conclusion: Endoscopic closure is seen as the first method for treating Stapfer type I perforations in the early phase, and surgery is seen as a remedial method when local treatment was failed. The Stapfer type II to type IV perforations can recover by conservative treatment.

Three new cassane-type diterpenoids from Caesalpinia sinensis.

Three new cassane-type diterpenoids, namely, (4S)-6ß,12α,19-trihydroxy-cass-13(15)-en-16,12-olide (1), cass-13(15)-en-​16,12-olide (2), and 12α-hydroxy-cass-13(15)-en-16,12-olide (3), were isolated from the seeds of Caesalpinia sinensis. The structures of 1-3 were established by extensive spectroscopic analysis, and their absolute configurations were assigned by electronic circular dichroism (ECD) calculations. The inhibitory activities against PTP1B of the isolated compounds were evaluated. The results showed that compound 2 possessed PTP1B inhibitory activity with an IC50 value of 217.45 ± 36.4 µM.

Logistic regression analysis on influencing factors of health cognition in patients after percutaneous coronary intervention / 公共卫生与预防医学

Objective To investigate the status of health knowledge of patients after percutaneous coronary intervention (PCI) and its influencing factors, so as to provide a scientific basis for formulating targeted health education programs. Methods Using a self-designed questionnaire on health cognition after PCI, 433 patients with a history of PCI surgery at a Grade III, Class A hospital between January 2019 and December 2019 were selected as the research subjects to investigate their health cognition and the influencing factors by binary logistic regression analysis. Results The pass rate of health cognition of patients after PCI was 39%, and the total correct rate was 57.5%. The correct rate of drug knowledge and risk factors was the lowest (33.6% and 48.5%, respectively). Binary logistic regression showed that gender, age and cardiac function classification were risk factors for health cognition of patients after PCI, while education level, occupation category and recurrence of discomfort after PCI were protective factors. Conclusion The level of health cognition of patients after PCI is low. Gender, age, education level, occupation category, cardiac function classification and recurrence of discomfort after PCI are independent influencing factors. When implementing health education, medical staff should comprehensively consider the formulation of individualized health education to help.

Meta-analysis on effect of Kushen Gel on high-risk cervical HPV infection / 中草药

Objective: To systematically evaluate the effectiveness of Kushen Gel in the treatment of cervical high-risk human papillomavirus (HR-HPV) infection. Methods: The Cochrane Library, PubMed, Embase, Medline, Wanfang, VIP, CNKI, and CBM databases were electronically searched to collect randomized controlled trials (RCTs) on Kushen Gel in the treatment of the cervical high-risk HPV infection from their inception to December 2019. Two researchers screened the articles, extracted the data, assessed the risk of bias of the eligble studies independently, and performed Meta-analysis with RevMan 5.3. Results: A total of 583 cases were included in five RCTs. The Meta-analysis showed that HR-HPV negative rate [RR = 1.38, 95% CI(1.20, 1.58), P 0.05). Conclusion: The therapeutic effect of applying Kushen Gel can increase the rate of clearance of HPV, reduce HPV-DNA load, and reduce postoperative complication. There is no evidence that the use of Kushen Gel after LEEP can reduce the recurrence rate of CIN2/CIN3. Due to the limitation of the quantity and quality of the included studies, the results need to be further verified and updated by more high-quality RCTs.

Is Chinese Medicine Injection Applicable for Treating Acute Lung Injury and Acute Respiratory Distress Syndrome? A Systematic Review and Meta-analysis of Randomized Controlled Trials / 中国结合医学杂志

OBJECTIVE@#To assess the efficacy and safety of Chinese medicine injection (CMI) for treating acute lung injury/acute respiratory distress syndrome (ALI/ARDS).@*METHODS@#Randomized controlled trials (RCTs) were identified by searching 3 English databases and 4 Chinese databases from their inceptions until February 2019. The Cochrane Handbook was used to evaluate risk of bias in the included studies. Data analysis was conducted using RevMan 5.3.3 software.@*RESULTS@#A total of 19 eligible RCTs involving 1,334 participants was included in this systematic review and meta-analysis. The main meta-analysis showed that CMI combined with conventional therapy (CT) was more effective than CT alone in reducing the acute physiology and chronic health evaluation (APACHE) H score [mean difference (MD): -1.74 points, 95% confidence interval (CI): -2.77 to -0.71, I@*CONCLUSIONS@#CMI as an adjuvant therapy showed great potential benefits for the treatment of ALI/ARDS. However, we could not make a definite conclusion due to low quality of included studies and uncertain security. Future studies should focus on improving research design, especially in blindness and placebo. The reporting of adverse events was also needed.

The evolving conception and practice of acupuncture-moxibustion / 针灸推拿医学（英文版）

With the emergence of the modified forms of acupuncture-moxibustion such as dry needle,the discipline acupuncture-moxibustion faces significant opportunities and challenges.The concept and treatment of acupuncture-moxibustion need to combine with modern medicine to consolidate the effectiveness and apply the research results to guide clinical treatment.By reviewing the brief history of acupuncture-moxibustion in the Western countries and summarizing the definitions,this article was to propose the trend and development strategies of this discipline in the future.

Therapeutic Observation of Acupoint Injection at Fenglong (ST 40) with Promethazine for Posterior Circulation Ischemic Vertigo Due to Turbid Phlegm Obstructing the Middle / 上海针灸杂志

Objective To observe the clinical efficacy of acupoint injection at Fenglong (ST 40) with Promethazine in treating posterior circulation ischemic vertigo (PCIV) due to turbid phlegm obstructing the middle.Method Sixty-two patients with PCIV due to turbid phlegm obstructing the middle were randomized into a treatment group and a control group, 31 cases each. The two groups both received intravenous infusion of Vinpocetine injection, based on which, the treatment group was intervened by injection at Fenglong (ST 40) with Promethazine, while the control group was given gluteal intramuscular injection of Promethazine. The traditional Chinese medicine (TCM) syndrome score and Dizziness Handicap Inventory (DHI) were observed for the two groups before and after the treatment, and the clinical efficacies were also compared.Result The TCM syndrome and DHI scores were significant changed after the intervention in both groups (P<0.05). After the treatment, the TCM syndrome and DHI scores in the treatment group were significantly different from those in the control group (P<0.05). The total effective rate was 93.5％ in the treatment group versus 80.6％ in the control group, and the between-group difference was statistically significant (P<0.05).Conclusion Injection at Fenglong (ST 40) with Promethazine is an effective method in treating PCIV due to turbid phlegm obstructing the middle.

Changes of CD3 +CD8 +T cells and its relationships with markers of inflammation and humoral immu-nity in children with severe bacterial meningitis / 中国小儿急救医学

Objective To explore the role of CD3 +CD8 +T cells in children with severe bacterial meningitis at early stage by studying the changes of CD3 +CD8 +T cells and its relationships with markers of inflammation and humoral immunity. Methods Thirty-nine cases aged ≤14 years old were enrolled in this retrospective study,who were admitted in our PICU from Aug 1. 2014 to Dec 31. 2015 with diagnosis of se-vere bacterial meningitis. The patients were divided into 2 groups according to the changes of CD3 +CD8 + T cells:group A:normal or elevated(≥190/mm3,n=22),group B:decreased( 80% was more than 7 cases(31. 8%) in group A,there were significant differences(P<0. 05). Fourteen cases(82. 3%) with glucose concentration lower than 2. 0 mmol/L in group B was more than 11 cases (50. 0%) in group A,the difference was significant(P<0. 05). Conclusion CD3 +CD8 +T cells might be suppressed in children with severe bacterial meningitis at early stage,and might associate to the damaging de-gree of brain,inflammation reaction and prognosis in those patients,which should be helpful in using of im-mune regulators for children with severe bacterial meningitis.

Value of acupuncture in life science / 第二军医大学学报

Acupuncture is the leading discipline of traditional Chinese medicine and undertakes the communication between traditional Chinese medicine and other natural sciences. With the rapid development of life science and technology and the exponential growth of knowledge, traditional acupuncture faces significant opportunities and challenges. In the context of great development of life sciences, we have re-recognized acupuncture, and we believe that acupuncture effects have corresponding material basis and regulatory mechanisms. The full embodiment of life science in the definition of acupuncture will contribute to the synergetic development of clinical and basic acupuncture research. In this paper, we discussed the value of acupuncture in life science based on the concepts of acupuncture and material basis of acupuncture effects.

Genetic variation analysis of Nsp2, ORF5 and ORF3 of PRRSV SC-GY strain from Sichuan province / 中国兽医学报

To monitor genetic variation of porcine reproductive and respiratory syndrome virus (PRRSV),RT-PCR was used to identify a sample suspected of PRRSV infection.A PRRSV named SC-GY strain was obtained,and its Nsp2,ORF5 and ORF3 genes were used for sequence alignment and phylogenetic tree construction.The results showed that SC-GY strain is highly pathogenic PRRSV American variant strains with Nsp2 gene discontinuous deletion of 30 amino acids,ORF3 gene aa17 a serine (S) insert.Comparing to VR2332,CH-1a,JXA1,HUN4,NADC30,HENAN-XINX and SC2012,the Nsp2,ORF5 and ORF3 of SC-GY shared 70.3％-97.9％,82.4％-97.6％ and 83.1％-98.2％ of nucleotide similarity,and 62.3％-96.3％,78.0％-95.7％ and 81.6％-96.5％ of deduced amino acid similarity;and compared to LV they shared only 18.9％,60.8％ and 63.7％ of nucleotide similarity,and 14.0％,54.9％ and 57.2％ of deduced amino acid similarity.The phylogenetic tree revealed that the SC-GY formed independent small branches although it belonged to the same subgroup as highly pathogenic PRRSV strains.The results showed that in high frequency live vaccine immunization of currently PRRSV,the gene of PRRSV epidemic strain is still in constant variation.Vaccination of live PRRSV vaccines should be reduced and surveillance of PRRSV strains should be enhanced.

Use of rhenium-188 for in vivo imaging and treatment of human cervical cancer cells transfected with lentivirus expressing sodium iodide symporter.

Although survival rates for cervical cancer have improved, they need further improvement in patients with distant metastases. The sodium iodine symporter (NIS) gene has often been used in cancer therapy and imaging. We examined the therapeutic effects of rhenium-188 (188Re) in a cervical cancer xenograft model expressing the NIS gene under the control of the tumor-specific human telomerase reverse transcriptase (hTERT) promoter. We constructed two recombinant lentiviral vectors expressing enhanced green fluorescent protein (eGFP) or the NIS gene driven by the hTERT promoter. To determine the tumor-specific transcriptional activity of the hTERT promoter, the eGFP-expressing vector was stably transfected into tumor cells and normal cells. A cervical cancer HeLa cell line stably expressing NIS (HeLa-TERTNIS) was created and examined in a similar way. HeLa and HeLa-TERTNIS tumor xenografts were transplanted in nude mice, and in vivo 188Re distribution was measured using micro-SPECT/CT imaging. The therapeutic effects of 188Re were assessed over 21 days on the basis of tumor volume and the immunohistochemical findings of excised tumors. eGFP expression controlled by the hTERT promoter was substantially higher in the tumor cells than normal cells. Quantitative PCR and western blotting confirmed that HeLa-TERTNIS cells expressed high levels of NIS mRNA and protein, respectively. Further, 188Re uptake and accumulation were significantly higher in HeLa-TERTNIS cells and xenografts than HeLa cells and xenografts. In vitro and in vivo, 188Re significantly reduced the survival of HeLa-TERTNIS cells and inhibited the growth of HeLa-TERTNIS xenografts, respectively. Immunohistochemical staining showed that HeLa-TERTNIS xenograft tumors expressed higher levels of NIS and caspase-3 and lower levels of Ki-67 than HeLa xenograft tumors. Our findings indicated that hTERT promoter-driven expression of the NIS gene in HeLa cells led to 188Re uptake and therapeutic effects. Thus, NIS-based gene therapy and imaging using the hTERT promoter and 188Re may be possible.

Fragile Sites of 'Valencia' Sweet Orange (Citrus sinensis) Chromosomes Are Related with Active 45s rDNA.

Citrus sinensis chromosomes present a morphological differentiation of bands after staining by the fluorochromes CMA and DAPI, but there is still little information on its chromosomal characteristics. In this study, the chromosomes in 'Valencia' C. sinensis were analyzed by fluorescence in situ hybridization (FISH) using telomere DNA and the 45S rDNA gene as probes combining CMA/DAPI staining, which showed that there were two fragile sites in sweet orange chromosomes co-localizing at distended 45S rDNA regions, one proximally locating on B-type chromosome and the other subterminally locating on D-type chromosome. While the chromosomal CMA banding and 45S rDNA FISH mapping in the doubled haploid line of 'Valencia' C. sinensis indicated six 45S rDNA regions, four were identified as fragile sites as doubled comparing its parental line, which confirmed the cytological heterozygosity and chromosomal heteromorphisms in sweet orange. Furthermore, Ag-NOR identified two distended 45S rDNA regions to be active nucleolar organizing regions (NORs) in diploid 'Valencia' C. sinensis. The occurrence of quadrivalent in meiosis of pollen mother cells (PMCs) in 'Valencia' sweet orange further confirmed it was a chromosomal reciprocal translocation line. We speculated this chromosome translocation was probably related to fragile sites. Our data provide insights into the chromosomal characteristics of the fragile sites in 'Valencia' sweet orange and are expected to facilitate the further investigation of the possible functions of fragile sites.

Changes in blood CD4CD25regulatory T cells in children with severe purulent meningitis / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To preliminarily study the changes in CD4CD25regulatory T cells (Tregs) in children with severe purulent meningitis at the early stage and its possible implications.</p><p><b>METHODS</b>A retrospective analysis was performed on the clinical data of 39 children with severe purulent meningitis who were admitted to the pediatric intensive care unit from August 2014 to December 2015. According to whether Tregs count was decreased within 12 hours of hospitalization (considering Tregs count <410/mmas decreased), they were divided into two groups: decrease group and non-decrease group. The associations between the changes in Tregs cells and the clinical manifestations, laboratory marker levels, and prognosis were analyzed.</p><p><b>RESULTS</b>Of the 39 cases, 13 (33%) showed a decrease in the proportion of Tregs cells (<31%) and 18 (46%) showed a decrease in the absolute Tregs cell count (<410/mm). Four deaths were all in the Tregs decrease group. Compared with the non-decrease group, the decrease group showed a significantly higher proportion of children with a peripheral blood leukocyte count lower than the normal range and a significantly greater increase in the level of serum procalcitonin (P<0.05).</p><p><b>CONCLUSIONS</b>Tregs might be suppressed in children with severe purulent meningitis at the early stage. And its suppression could be related to the severer inflammation reaction and higher mortality in those patients.</p>

Study of a High-Yield Cellulase System Created by Heavy-Ion Irradiation-Induced Mutagenesis of Aspergillus niger and Mixed Fermentation with Trichoderma reesei.

The aim of this study was to evaluate and validate the efficiency of 12C6+ irradiation of Aspergillus niger (A. niger) or mutagenesis via mixed Trichoderma viride (T. viride) culturing as well as a liquid cultivation method for cellulase production via mixed Trichoderma reesei (T. reesei) and A. niger culture fermentation. The first mutagenesis approach was employed to optimize yield from a cellulase-producing strain via heavy-ion mutagenesis and high-throughput screening, and the second was to effectively achieve enzymatic hydrolysis of cellulase from a mixed culture of mutant T. viride and A. niger. We found that 12C6+-ion irradiation induced changes in cellulase biosynthesis in A. niger but had no effect on the time course of the synthesis. It is notable that the exoglucanases (CBH) activities of A. niger strains H11-1 and H differed (6.71 U/mL vs. 6.01 U/mL) and were significantly higher than that of A. niger mutant H3-1. Compared with strain H, the filter paper assay (FPA), endoglucanase (EG) and ß-glucosidase (BGL) activities of mutant strain H11-1 were increased by 250.26%, 30.26% and 34.91%, respectively. A mixed culture system was successfully optimized, and the best ratio of T. reesei to A. niger was 5:1 for 96 h with simultaneous inoculation. The BGL activity of the mixed culture increased after 72 h. At 96 h, the FPA and BGL activities of the mixed culture were 689.00 and 797.15 U/mL, respectively, significantly higher than those of monocultures, which were 408.70 and 646.98 U/mL for T. reesei and 447.29 and 658.89 U/mL for A. niger, respectively. The EG activity of the mixed culture was 2342.81 U/mL, a value that was significantly higher than that of monocultures at 2206.57 U/mL for T. reesei and 1727.62 U/mL for A. niger. In summary, cellulose production and hydrolysis yields were significantly enhanced by the proposed combination scheme.