Human liver cancer organoids: Biological applications, current challenges, and prospects in hepatoma therapy.

Liver cancer and disease are among the most socially challenging global health concerns. Although organ transplantation, surgical resection and anticancer drugs are the main methods for the treatment of liver cancer, there are still no proven cures owing to the lack of donor livers and tumor heterogeneity. Recently, advances in tumor organoid technology have attracted considerable attention as they can simulate the spatial constructs and pathophysiological characteristics of tumorigenesis and metastasis in a more realistic manner. Organoids may further contribute to the development of tailored therapies. Combining organoids with other emerging techniques, such as CRISPR-HOT, organ-on-a-chip, and 3D bioprinting, may further develop organoids and address their bottlenecks to create more practical models that generalize different tissue or organ interactions in tumor progression. In this review, we summarize the various methods in which liver organoids may be generated and describe their biological and clinical applications, present challenges, and prospects for their integration with emerging technologies. These rapidly developing liver organoids may become the focus of in vitro clinical model development and therapeutic research for liver diseases in the near future.

Afterglow Electrochemiluminescence from Nitrogen-Deficient Graphitic Carbon Nitride.

Almost all current electrochemiluminescent reagents require real-time electrochemical stimulation to emit light. Here, we report a novel electrochemiluminescent reagent, nitrogen-deficient graphitic carbon nitride (CNx), that can emit afterglow electrochemiluminescence (ECL) after cessation of electric excitation. CNx obtained by post-thermal treatment of graphitic carbon nitride (CN) with KSCN has a cyanamide group and a nitrogen vacancy, which created defects to trap electrically injected electrons. The trapped electrons can slowly release and react with coreactants to emit light with longevity. The cathodic afterglow ECL lasts for 70 s after pulsing the CNx nanosheet (CNxNS-1.6)-modified glassy carbon electrode at -1.0 V for 20 s in 2.0 M PBS containing 1 mM K2S2O8. The afterglow ECL mechanism is revealed by investigation of its influencing factors and ECL wavelength. The discovery of afterglow ECL may open a new doorway for new significant applications of the ECL technique and provide a deeper understanding of the structure-property relationships of CN.

DDX56 transcriptionally activates MIST1 to facilitate tumorigenesis of HCC through PTEN-AKT signaling.

Rationale: Hepatocellular carcinoma (HCC) is a primary malignancy of the liver that is the leading cause of cancer-related mortality worldwide. However, genetic alterations and mechanisms underlying HCC development remain unclear. Methods: Tissue specimens were used to evaluate the expression of DEAD-Box 56 (DDX56) to determine its prognostic value. Colony formation, CCK8, and EdU-labelling assays were performed to assess the effects of DDX56 on HCC proliferation. The in vivo role of DDX56 was evaluated using mouse orthotopic liver xenograft and subcutaneous xenograft tumor models. Dual-luciferase reporter, chromatin immunoprecipitation, and electrophoretic mobility shift assays were performed to examine the effect of DDX56 on the MIST1 promoter. Results: DDX56 expression in HCC tissues was elevated and this increase was strongly correlated with poor prognoses for HCC patients. Functionally, DDX56 promoted HCC cell proliferation both in vitro and in vivo, while mechanistically interacting with MECOM to promote HCC proliferation by mono-methylating H3K9 (H3K9me1) on the MIST1 promoter, leading to enhanced MIST1 transcription and subsequent regulation of the PTEN/AKT signaling pathway, which promotes HCC proliferation. More importantly, the PTEN agonist, Oroxin B (OB), blocked the DDX56-mediated PTEN-AKT signaling pathway, suggesting that treating HCC patients with OB may be beneficial as a therapeutic intervention. Furthermore, we observed that ZEB1 bound to DDX56 and transcriptionally activated DDX56, leading to HCC tumorigenesis. Conclusions: Our results indicated that the ZEB1-DDX56-MIST1 axis played a vital role in sustaining the malignant progression of HCC and identified DDX56 as a potential therapeutic target in HCC tumorigenesis.

Tumor Cell-Specific and Lipase-Responsive Delivery of Hydrogen Sulfide for Sensitizing Chemotherapy of Pancreatic Cancer.

The inability of small molecule drugs to diffuse into tumor interstitium is responsible for the relatively low effectiveness of chemotherapy. Herein, a hydrogen sulfide (H2S) gas-involved chemosensitization strategy is proposed for pancreatic cancer treatment by developing a tumor-specific lipase-responsive nanomedicine based on aptamer-conjugated DATS/Dox co-loaded PCL-b-PEO micelle (DA/D@Ms-A). After receptor-mediated endocytosis and subsequent digestion of PCL blocks by intracellular lipase, the nanomedicine releases Dox and DATS, which then react with intracellular glutathione to produce H2S. The cytotoxicity result indicates that H2S can enhance Dox chemotherapy efficiency owing to the synergetic therapeutic effect of Dox and H2S. Moreover, the nanomedicine is featured with well tumor penetration capability benefitting from the targeting ability of aptamers and high in vivo biocompatibility due to the high density of PEO and biodegradable PCL. The nanomedicine capable of synergetic gas-chemotherapy holds great potential for pancreatic cancer treatment.

Efficacy of Auricular Acupressure in Prevention and Treatment of Chemotherapy-Induced Nausea and Vomiting in Patients with Cancer: A Systematic Review and Meta-Analysis.

BACKGROUND: More than 40% of patients with cancer have reported that chemotherapy-induced nausea and vomiting (CINV) remained the most debilitating side effects of treatment even in the era of new antiemetics. OBJECTIVE: The purpose of this review was to systematically evaluate the clinical effect of auricular acupressure (AA) in prevention and treatment of chemotherapy-induced nausea and vomiting. METHODS: The following databases were searched: PubMed, Cochrane Library, EMBASE, the Web of Science, Chinese Biological Medicine (CBM), Chinese National Knowledge Infrastructure (CNKI), Wanfang, and VIP (from database inception to April 2020). Eligible randomized controlled trials of auricular acupressure in treating CINV were collected, including crossover randomized design study. The meta-analysis was carried out by RevMan software (5.3). RESULTS: Totally 19 RCTs with 1449 patients met the inclusion criteria. Compared with control groups, the relief efficiency of overall CINV was enhanced by AA combined with antiemetics (RR = 1.31, CI 1.22 to 1.41, p ≤ 0.001). Although the therapeutic effect on acute nausea and vomiting was not obvious, AA still played an important role in reducing delayed nausea and vomiting (delayed nausea frequency: RR = 0.68, CI -1.01 to -1.35, p ≤ 0.001; delayed vomiting frequency: RR = 0.91, CI -1.22 to -0.61, p ≤ 0.001). The likelihood of adverse reactions related to antiemetics was reduced by AA combined with antiemetics (RR = 0.62, CI 0.53 to 0.74, p ≤ 0.001). Statistically significant association was found between AA and incidence of constipation, diarrhea, and tiredness, while there was no statistically significant association between AA and abdominal distension or headache. CONCLUSION: Auricular acupressure supplementation benefited delayed chemotherapy-induced nausea and vomiting as well as constipation, diarrhea, and tiredness. AA alone or AA supplementation has little effect on acute nausea and acute vomiting. There is no conclusion on whether AA alone is superior to antiemetics in the management of delayed CINV. Further studies are needed to confirm the efficacy of auricular acupressure alone in delayed CINV and anticipatory CINV. The results of this review provided the basis for further research with more rigorous study designs, adequate sample sizes, and standardized implementation to confirm the efficacy of auricular acupressure.

Hybridizing Carbon-Based Dot-Capped Manganese Dioxide Nanosheets and Gold Nanoparticles as a Highly Sensitive Surface-Enhanced Raman Scattering Substrate.

Surface-enhanced Raman Scattering (SERS) is a sensitive and nondestructive technique that provides fingerprint structural information of molecules. Designing and constructing sensitive and stable SERS substrates is of great significance for the application of the technique. In this study, single-layer carbon-based dots (CDs) are used as capping agents to synthesize gold nanoparticles (AuNPs/CDs) and manganese dioxide nanosheets (MnO2/CDs), which are then hybridized through a simple cocentrifugation method. After the hybridization, the monodispersive AuNPs/CDs aggregate obviously into some clusters exhibiting strong SERS activity due to the electromagnetic "hot spots" effect, and the MnO2/CDs also show outstanding SERS activity due to the charge-transfer resonance effect. The obtained nanohybrids (MnO2/CDs/AuNPs) with robust chemical stability combine well with the electromagnetic enhancement of AuNPs/CDs and chemical enhancement of MnO2/CDs, leading to an ultrahigh enhancement factor of 3.9 × 108. Based on the novel SERS substrate, a sensitive and rapid sensing system for the detection of malachite green is developed, with a low detection limit of 1 × 10-9 M. This work provides a valuable model for designing and fabricating high-performance SERS substrates.

Tune the Fluorescence and Electrochemiluminescence of Graphitic Carbon Nitride Nanosheets by Controlling the Defect States.

The effects of defect states on the fluorescence (FL) and electrochemiluminescence (ECL) properties of graphite phase carbon nitride (g-CN) are systematically investigated for the first time. The g-CN nanosheets (CNNSs) obtained at different condensation temperatures are used as the study models. It can be found that all the CNNSs have two kinds of defect states, one is originated from the edge of CNNSs (labeled as CN-defect) and the other is attributed to the partially carbonization regions (labeled as C-defect). Both two kinds of defect states substantially affect the luminescent properties of CNNSs. Both the FL and ECL signals of CNNSs contain a band gap emission and two defect emissions. For the FL of CNNSs, decreasing the density of defect states can increase efficiently the FL quantum yield, while increasing the density of defect states can make the FL spectra red shift. For the ECL of CNNSs, increasing the density of CN-defect states and decreasing the density of C-defect states are greatly important to improve the ECL activity. This work provides a deep insight into the FL and ECL mechanisms of g-CN, and is of significance in tuning the FL and ECL properties of g-CN. Also, it will greatly promote the applications of CNNSs based on the FL and ECL properties.

Heparin versus 0.9% saline solution to maintain patency of totally implanted venous access ports in cancer patients: A systematic review and meta-analysis.

AIM: The use of heparin and 0.9% saline solution is always controversial for central venous catheters. However, there is no systematic review or guideline about whether saline solution can replace heparin solution in adult cancer patients with totally implantable venous access ports (TIVAPs). The purpose of this review is to evaluate whether saline solution can replace heparin saline to lock TIVAPs. METHODS: The following databases were searched: PubMed, the Cochrane Library, Web of Science, Embase, CINAHL and Ovid (January 1, 1982, and February 21, 2020). All statistical analyses of the meta-analysis were completed using the Review Manager 5.3. RESULTS: A total of 201 studies were identified from these databases after initial review, and four studies met inclusion criteria, including 2652 cases. There was little heterogeneity among the included studies (I2 < 30%), and all analyses were conducted by the fixed-effects model. The total complications, catheter occlusions, catheter-related bloodstream infections and other complication rates in the heparin solution group were higher than in the saline solution group. In the subgroup analysis of heparin concentration, total complication rates in the saline solution group were higher than with 50 U of heparin and lower than with 100 U of heparin. However, the differences in these complications were small, and no significant difference was observed (all P > 0.05). CONCLUSIONS: Based on existing clinical studies, we recommend that saline solution can replace 50 or 100 U/ml of heparin as a safe and effective flush solution for TIVAPs.

Prolonging the flush-lock interval of totally implantable venous access ports in patients with cancer: A systematic review and meta-analysis.

BACKGROUND: Recently, some studies have shown that prolonging flush interval is safe and feasible for patients who complete chemotherapy. However, there is no consensus about the optimal flush interval for those patients. OBJECTIVE: The purpose of this review was to evaluate whether the flush interval could be prolonged based on monthly interval for regular maintenance and to explore the optimal flush interval. DATA SOURCES: We searched the following databases for articles published between 1 January 1982 and 21 February 2020: PubMed, Cochrane Library, Web of Science, EMBASE, CINAHL, and Ovid. STUDY ELIGIBILITY CRITERIA: Randomized controlled trials, retrospective and prospective cohort studies of flush interval less than 4 weeks versus longer than 4 weeks for patients who completed chemotherapy, were included. RESULTS: Two reviewers extracted information and assessed the quality of the articles independently. In total, 389 articles were retrieved, and 4 studies including 862 cases fulfilled the inclusion criteria. There was no statistical heterogeneity (I2 = 0, p > 0.05) among the included studies. Hence, the fixed-effects model was used for the meta-analysis. The meta-analysis showed that the total complication rate associated with longer than 4-week interval was higher than that associated with less than 4-week interval. Nevertheless, there was no significant difference between the two groups (7.2% vs 7.6%, p = 0.83). Moreover, the meta-analysis showed that the total complication and catheter occlusion rates associated with the 4-week interval were higher than those associated with the 8-week interval. However, there was no significant difference between the two groups (total complications: 11.4% vs 9.5%, p = 0.68; catheter occlusions: 4.9% vs 4.1%, p = 0.89). LIMITATIONS: Only four non-randomized controlled studies were included, and the outcomes of the included studies were reported incompletely. CONCLUSION: Extending the flush interval to longer than 4 weeks is safe and feasible. Based on previous studies, extending the flush interval to 8 weeks might not increase the incidence of total complications and catheter occlusions. However, there is no conclusion on whether the flush interval could be extended to 3 months or longer.

Ultra-high quantum yield ultraviolet fluorescence of graphitic carbon nitride nanosheets.

Graphitic carbon nitride (g-CN) nanosheets (CNNs) with an ultra-high quantum yield (80.1%) ultraviolet fluorescence (FL) were prepared. The effects of the lateral size and the polymerization temperature on the optical properties of CNNs have been studied. The ultraviolet FL was proved to have originated from the isolated melem units according to the density functional theory calculation and mass spectra. The obtained CNNs are further used as a pH probe due to the dependence of the FL signal on the pH of the solution.

Gas-Mediated Cancer Bioimaging and Therapy.

Gas-involving cancer theranostics have attracted considerable attention in recent years due to their high therapeutic efficacy and biosafety. We have reviewed the recent significant advances in the development of stimuli-responsive gas releasing molecules (GRMs) and gas nanogenerators for cancer bioimaging, targeted and controlled gas therapy, and gas-sensitized synergistic therapy. We have focused on gases with known anticancer effects, such as oxygen (O2), carbon monoxide (CO), nitric oxide (NO), hydrogen sulfide (H2S), hydrogen (H2), sulfur dioxide (SO2), carbon dioxide (CO2), and heavy gases that act via the gas-generating process. The GRMs and gas nanogenerators for each gas have been described in terms of the stimulation method, followed by their applications in ultrasound and multimodal imaging, and finally their primary and synergistic actions with other cancer therapeutic modalities. The current challenges and future possibilities of gas therapy and imaging vis-à-vis clinical translation have also been discussed.

Spontaneous Cancers, But Not Many Induced Ones in Animals, Resemble Semi-New Organisms that Possess a Unique Programmed Cell Death Mode Different from Apoptosis, Senescent Death, Necrosis and Stress-Induced Cell Death.

There are four basic cell death modes in animals, i.e. physiological senescent death (SD) and apoptosis as well as pathological necrosis and stress-induced cell death (SICD). There have been numerous publications describing "apoptosis" in cancer, mostly focused on killing cancer cells using radio- or chemo-therapy, with few on exploring how cancer cells die naturally without such treatments. Spontaneous benign or malignant neoplasms are immortal and autonomous, but they still retain some allegiance to their parental tissue or organ and thus are still somewhat controlled by the patient's body. Because of these properties of immortality, semi-autonomy, and semi-allegiance to the patient's body, spontaneous tumors have no redundant cells and resemble "semi-new organisms" parasitizing the patients, becoming a unique tissue type possessing a hitherto unannotated cell death mode besides SD, apoptosis, necrosis and SICD. Particularly, apoptosis aims to expunge redundant cells, whereas this new mode does not. In contrast to spontaneous tumors, many histologically malignant tumors induced in experimental animals, before they reach an advanced stage, regress after withdrawal of the inducer. This mortal and non-autonomous nature disqualifies these animal lesions as authentic neoplasms and as semi-new organisms but makes them a good tissue type for apoptosis studies. Ruminating over cell death in spontaneous cancers and many inauthentic tumors induced in animals from these new slants makes us realize that "whether cancer cells undergo apoptosis" is not an easy question with a simple answer. Our answer is that cancer cells have an uncharacterized programmed cell death mode, which is not apoptosis.

Evaluating the Remote Control of Programmed Cell Death, with or without a Compensatory Cell Proliferation.

Organisms and their different component levels, whether organelle, cellular or other, come by birth and go by death, and the deaths are often balanced by new births. Evolution on the one hand has built demise program(s) in cells of organisms but on the other hand has established external controls on the program(s). For instance, evolution has established death program(s) in animal cells so that the cells can, when it is needed, commit apoptosis or senescent death (SD) in physiological situations and stress-induced cell death (SICD) in pathological situations. However, these programmed cell deaths are not predominantly regulated by the cells that do the dying but, instead, are controlled externally and remotely by the cells' superior(s), i.e. their host tissue or organ or even the animal's body. Currently, it is still unclear whether a cell has only one death program or has several programs respectively controlling SD, apoptosis and SICD. In animals, apoptosis exterminates, in a physiological manner, healthy but no-longer needed cells to avoid cell redundancy, whereas suicidal SD and SICD, like homicidal necrosis, terminate ill but useful cells, which may be followed by regeneration of the live cells and by scar formation to heal the damaged organ or tissue. Therefore, "who dies" clearly differentiates apoptosis from SD, SICD and necrosis. In animals, apoptosis can occur only in those cell types that retain a lifelong ability of proliferation and never occurs in those cell types that can no longer replicate in adulthood. In cancer cells, SICD is strengthened, apoptosis is dramatically weakened while SD has been lost. Most published studies professed to be about apoptosis are actually about SICD, which has four basic and well-articulated pathways involving caspases or involving pathological alterations in the mitochondria, endoplasmic reticula, or lysosomes.

While it is not deliberate, much of today's biomedical research contains logical and technical flaws, showing a need for corrective action.

Biomedical research has advanced swiftly in recent decades, largely due to progress in biotechnology. However, this rapid spread of new, and not always-fully understood, technology has also created a lot of false or irreproducible data and artifacts, which sometimes have led to erroneous conclusions. When describing various scientific issues, scientists have developed a habit of saying "on one hand… but on the other hand…", because discrepant data and conclusions have become omnipresent. One reason for this problematic situation is that we are not always thoughtful enough in study design, and sometimes lack enough philosophical contemplation. Another major reason is that we are too rushed in introducing new technology into our research without assimilating technical details. In this essay, we provide examples in different research realms to justify our points. To help readers test their own weaknesses, we raise questions on technical details of RNA reverse transcription, polymerase chain reactions, western blotting and immunohistochemical staining, as these methods are basic and are the base for other modern biotechnologies. Hopefully, after contemplation and reflection on these questions, readers will agree that we indeed know too little about these basic techniques, especially about the artifacts they may create, and thus many conclusions drawn from the studies using those ever-more-sophisticated techniques may be even more problematic.

Effects of C-Related Dangling Bonds and Functional Groups on the Fluorescent and Electrochemiluminescent Properties of Carbon-Based Dots.

Single-layer carbon-based dots (SCDs) were chosen as a model to investigate the effect of the C-related dangling bonds with spin S=1/2 and functional groups on the electrochemiluminescent (ECL) and fluorescent (FL) properties of CDs. The C-related dangling bonds and functional groups of SCDs were tuned by chemical reduction with NaBH4 . There have several main findings via investigating the ECL and FL properties of SCDs before and after the chemical reduction. First, the FL and ECL of CDs are highly dependent on their concentration, and luminescent resonance energy transfer is observed in ECL studies when the concentration of CDs is high. Second, the ECL activity of CDs is greatly enhanced as the C-related dangling bonds increase, proving that the ECL of CDs originates from the C-related dangling bonds. Third, the FL of CDs is the synthesis of the inner FL originated from the contained isolated sp2 units and the defect FL from the C-related dangling bonds. The inner FL of CDs is enhanced greatly by removing the carboxyl groups, while the defect FL is increased slightly due to the increased C-related dangling bonds. We believe this study would promote our understanding in the ECL and FL mechanisms of CDs, advancing the applications of CDs based on their ECL and FL properties.

Transcriptional-Readthrough RNAs Reflect the Phenomenon of "A Gene Contains Gene(s)" or "Gene(s) within a Gene" in the Human Genome, and Thus Are Not Chimeric RNAs.

Tens of thousands of chimeric RNAs, i.e., RNAs with sequences of two genes, have been identified in human cells. Most of them are formed by two neighboring genes on the same chromosome and are considered to be derived via transcriptional readthrough, but a true readthrough event still awaits more evidence and trans-splicing that joins two transcripts together remains as a possible mechanism. We regard those genomic loci that are transcriptionally read through as unannotated genes, because their transcriptional and posttranscriptional regulations are the same as those of already-annotated genes, including fusion genes formed due to genetic alterations. Therefore, readthrough RNAs and fusion-gene-derived RNAs are not chimeras. Only those two-gene RNAs formed at the RNA level, likely via trans-splicing, without corresponding genes as genomic parents, should be regarded as authentic chimeric RNAs. However, since in human cells, procedural and mechanistic details of trans-splicing have never been disclosed, we doubt the existence of trans-splicing. Therefore, there are probably no authentic chimeras in humans, after readthrough and fusion-gene derived RNAs are all put back into the group of ordinary RNAs. Therefore, it should be further determined whether in human cells all two-neighboring-gene RNAs are derived from transcriptional readthrough and whether trans-splicing truly exists.

Probably less than one-tenth of the genes produce only the wild type protein without at least one additional protein isoform in some human cancer cell lines.

To estimate how many genes produce multiple protein isoforms, we electrophoresed proteins from MCF7 and MDA-MB231 (MB231) human breast cancer cells in SDS-PAGE and excised narrow stripes of the gel at the 48kD, 55kD and 72kD. Proteins in these stripes were identified using liquid chromatography and tandem mass spectrometry. A total of 765, 750 and 679 proteins from MB231 cells, as well as 470, 390 and 490 proteins from MCF7 cells, were identified from the 48kD, 55kD and 72kD stripes, respectively. We arbitrarily allowed a 10% technical variation from the proteins' theoretical molecular mass (TMM) and considered those proteins with their TMMs within the 43-53 kD, 49-61 kD and 65-79 kD ranges as the wild type (WT) expected from the corresponding stripe, whereas those with a TMM above or below this range as a smaller- or larger-group, respectively. Only 263 (34.4%), 269 (35.9%) and 151 (22.2%) proteins from MB231 cells and 117 (24.9%), 135 (34.6%) and 130 (26.5%) proteins from MCF7 cells from the 48kD, 55kD and 72kD stripes, respectively, belonged to the WT, while the remaining majority belonged to the smaller- or larger-groups. Only about 3-16%, on average about 10% regardless of the stripe and cell line, of the proteins appeared in only one stripe and within the WT range, while the remaining preponderance appeared also in additional stripe(s) or had a larger or smaller TMM. We conclude that few (fewer than 10%) of the human genes produce only the WT protein without additional isoform(s).

Circulating Tumor Cells: Moving Biological Insights into Detection.

Circulating tumor cells (CTCs) have shown promising potential as liquid biopsies that facilitate early detection, prognosis, therapeutic target selection and monitoring treatment response. CTCs in most cancer patients are low in abundance and heterogeneous in morphological and phenotypic profiles, which complicate their enrichment and subsequent characterization. Several methodologies for CTC enrichment and characterization have been developed over the past few years. However, integrating recent advances in CTC biology into these methodologies and the selection of appropriate enrichment and characterization methods for specific applications are needed to improve the reliability of CTC biopsies. In this review, we summarize recent advances in the studies of CTC biology, including the mechanisms of their generation and their potential forms of existence in blood, as well as the current CTC enrichment technologies. We then critically examine the selection of methods for appropriately enriching CTCs for further investigation of their clinical applications.

Cervical Cancers Manifest a High Rate of Infection by a High-Risk Human Papilloma Virus Subtype but a Very Low Rate of Infection by a Low-Risk Subtype in the Guiyang District of China.

The prevalence of infection by different genotypes of human papillomavirus (HPV) varies among different geographic areas. We studied the prevalence of infection by 21 HPV genotypes in cervical tissue specimens from 4213 women in the Guiyang district, that is located in the southwest of China and is dominated by minor ethnicities of Chinese, and 2074 cases in our cohort had pathological diagnosis available. The overall infection rate was 36.98%. Most (72.08%) infectors were positive for only one HPV subtype, with the remaining being cases infected by two or more subtypes. Infections by the HPV subtypes 16, 52 and 58 were the most prevalent, having rates of 34.66%, 16.03%% and 15.53%, respectively. The most common cervical lesions in HPV infections were genital warts, cervical cancer (CC) and cervical intraepithelial neoplasia (CIN). Age and age at first sexual activity were independent risk factors for HPV infections that in turn cause certain cervical lesions. Intriguingly, while 94.90% of the CC patients were infected by oncogenically high-risk (HR) HPV subtypes, only 2.75% and 2.29% of these patients were infected by oncogenically low-risk (LR) subtypes or other-subtypes with their oncogenicity unclear. The rates of infection by LR-HPVs and other-HPVs were also low, being 4.63% and 6.76%, respectively, in the patients with CIN that is a precursor lesion of CC, lower than the 8.54% and 18.20%, respectively, in the women without a cervical lesion. Our data provides an important foundation for prevention, diagnosis and treatment of HPV infection in Guiyang district and suggests that development of vaccines for prevention and treatment of CC in this area should first target the HPV subtypes 16, 52 and 58, but not subtype 18 as for many other places. It deserves study whether infections by certain LR-HPVs and other-HPVs may serve as attenuated live vaccines for prevention of CC.

Establishment of a Simple and Quick Method for Detecting Extended-Spectrum ß-Lactamase (ESBL) Genes in Bacteria.

Extended-spectrum ß-lactamase (ESBL) genes that render bacteria resistant to antibiotics are commonly detected using phenotype testing, which is time consuming and not sufficiently accurate. To establish a better method, we used phenotype testing to identify ESBL-positive bacterial strains and conducted PCR to screen for TEM (named after the patient Temoneira who provided the first sample), sulfhydryl reagent variable (SHV), cefotaxime (CTX)-M-1, and CTX-M-9, the 4 most common ESBL types and subtypes. We then performed multiplex PCR with 1 primer containing a biotin and hybridized the PCR products with gene-specific probes that were coupled with microbeads and coated with a specific fluorescence. The hybrids were linked to streptavidin-R-phycoerythrins (SA-PEs) and run through a flow cytometer, which sorted the fluorescently dyed microbeads and quantified the PEs. The results from single PCR, multiplex PCR, and cytometry were consistent with each other. We used this method to test 169 clinical specimens that had been determined for phenotypes and found 154 positive for genotypes, including 30 of the 45 samples that were negative for phenotypes. The CTX-M genotype tests alone, counting both positive and negative cases, showed 99.41% (168/169) consistency with the ESBL phenotype test. Thus, we have established a multiplex-PCR system as a simple and quick method that is high throughput and accurate for detecting 4 common ESBL types and subtypes.