Identification and validation of the surface proteins FIBG, PDGF-ß, and TGF-ß on serum extracellular vesicles for non-invasive detection of colorectal cancer: experimental study.

OBJECTIVES: The absence of non-invasive biomarkers for the early diagnosis of colorectal cancer (CRC) has contributed to poor prognosis. Extracellular vesicles (EVs) have emerged as promising candidates for cancer monitoring using liquid biopsy. However, the complexity of EVs isolation procedures and absence of clear targets for detecting serum-derived EVs have hindered the clinical application of EVs in early CRC diagnosis. METHODS: In the discovery phase, we conducted a comprehensive 4D-DIA proteomic analysis of serum-derived EVs samples from 37 individuals, performing an initial screening of EVs surface proteins. In the technical validation phase, we developed an extraction-free CRC-EVArray microarray to assess the expression of these potential EVs surface proteins in a multicenter study comprising 404 individuals. In the application phase, we evaluated the diagnostic efficacy of the CRC-EVArray model based on machine-learning algorithms. RESULTS: Through 4D-DIA proteomic analysis, we identified 7 potential EVs surface proteins showing significantly differential expression in CRC patients compared to healthy controls. Utilizing our developed high-throughput CRC-EVArray microarray, we further confirmed the differential expression of 3 EVs surface proteins, FIBG, PDGF-ß and TGF-ß, in a large sample population. Moreover, we established an optimal CRC-EVArray model using the NNET algorithm, demonstrating superior diagnostic efficacy with an AUC of 0.882 in the train set and 0.937 in the test set. Additionally, we predicted the functions and potential origins of these EVs-derived proteins through a series of multi-omics approaches. CONCLUSIONS: Our systematic exploration of surface protein expression profiles on serum-derived EVs has identified FIBG, PDGF-ß, and TGF-ß as novel diagnostic biomarkers for CRC. And the development of CRC-EVArray diagnostic model based on these findings provided an effective tool for the large-scale CRC screening, thus facilitating its translation into clinical practice.

Identifying the Unmet Supportive Care Needs, with Concomitant Influencing Factors, in Family Caregivers of Cancer Patients in China.

OBJECTIVE: The objective of the study is to assess the unmet needs of cancer caregivers and to identify the possible predictors of their supportive care needs in China. METHODS: This multicenter, cross-sectional study enrolled 449 cancer patients' family caregivers' dyads. Patients provided general information and Karnofsky performance status (KPS); caregivers provided general information and completed a survey of Chinese version of the Supportive Care Needs Survey-Partners and Caregivers Scale. The independent samples t-test, one-way analysis of variance, and multiple stepwise regression were used to analyze the factors that influence the needs of caregivers. RESULTS: A proportion of caregivers who had no needs were 5.6%. A proportion of caregivers with ≥ 5 moderate or high unmet needs and with ≥ 10 moderate or high unmet needs were 77.7% and 63.2%, respectively. Healthcare services and information needs and communication and relationship needs were the most prominent areas of caregivers' unmet needs. The item "Finding out about financial support and government benefits for you and/or the person with cancer" was the highest level of unmet needs at 78.6%. The level of unmet needs was related to the patient's physical function (KPS score), caregiver's educational levels, financial burden of healthcare, as well as the level of burden related to caregiving (working status, caring for others, caregiving experience, and total caregiving time). CONCLUSIONS: The level of unmet needs of family caregivers of cancer patients in China was higher. In clinical practice, more attention should be paid to family caregivers who take care of the patient with poor physical function, those who are highly educated, faced with higher financial burden of healthcare, and are currently working, as well as those who need to take care of others, spend more time caregiving, and have no caregiving experience.

Hepatitis B Virus Might Be Sensed by STING-Dependent DNA Sensors and Attenuates the Response of STING-Dependent DNA Sensing Pathway in Humans with Acute and Chronic Hepatitis B Virus Infection.

DNA-dependent activator of interferon regulatory factors (DAIs), interferon gamma inducible protein 16 (IFI16), DEAD-box polypeptide 41 (DDX41), DNA-dependent protein kinase (DNA-PK), meiotic recombination 11 homolog A (MRE11), and cyclic GMP-AMP synthase (cGAS) have been identified as intracellular STING-dependent DNA sensors in recent years. Studies have shown that the DNA sensor-STING-interferon (IFN)-ß pathway plays an important role in the defense against intracellular invasion of many DNA viruses. However, the intracellular recognition of hepatitis B virus (HBV) DNA by DNA sensors is still largely unclear. In this study, we aimed to determine whether the DNA sensor-STING pathway in peripheral blood mononuclear cells (PBMCs) can be activated by acute and chronic HBV infections in humans. We first evaluated the expression of these DNA sensors in PBMCs of acute and chronic HBV-infected patients by quantitative real-time polymerase chain reaction. We next compared the expression of the upregulated DNA sensor between monocytes and nonmonocytes to find its cellular source. Finally, by in vitro stimulation, we analyzed the IFN-ß response of the DNA sensor-STING pathway in PBMCs and monocytes from chronic HBV-infected patients. The results showed that IFI16, DDX41, MRE11, and the adaptor STING were upregulated in chronic HBV-infected patients, whereas only IFI16 was upregulated in acute HBV-infected patients. However, IFN-ß expression was not changed in PBMCs from acute and chronic HBV-infected patients. We next found IFI16 was mainly expressed in monocytes of acute and chronic hepatitis B patients. Finally, by stimulation of monocytes with VACV ds 70mer, a ligand for IFI16, we confirmed the attenuated response of the IFI16-STING pathway. Taken together, our results suggest that HBV might be sensed by DNA sensors in PBMCs of acute and chronic HBV-infected patients, and meanwhile HBV infection attenuates the response of the DNA sensor-STING pathway in PBMCs and monocytes, which may facilitate the persistence of HBV infection.

Prevalence of Hepatitis B Virus Infection in Shenzhen, China, 2015-2018.

China has nearly 10% of the general HBV carrier population in the world; this infection is the most common cause of chronic liver disease. Understanding HBV epidemiology is essential for future infection control, evaluation, and treatment. This study determined the prevalence of HBV infection in Shenzhen by serological testing and analysis in 282,166 HBV screening cases for the following: HBcAb, indicative of previous HBV infection; HBsAg, indicative of chronic (current) infection; HBsAb, indicative of immunity from vaccination; and 34,368 HBV etiological screening cases for HBV-DNA, indicative of virus carriage, in which 1,204 cases were genotyped and mutation analyzed for drug-resistance evaluation. Shenzhen was a highly endemic area of HBV throughout the study period (prevalence 9.69%). HBV infections were almost entirely in the 20 and older age groups with a male-to-female ratio of 1.16:1 which is approximately the same as the male-to-female ratio of the general population in China. However, only 71.25% of the general population retained HBV immune protection. Genotype B and C were identified as the most common agents; recombinant B/C and B/D also existed; some cases, however, could not be genotyped. NAs resistant mutation occurrence patterns were multitudinous; single mutation patterns of rtM204I/V and rtL180M occurrences accounted for majority, followed by the combinational mutation pattern L180M + M204I/V. Drug-resistance was prevalent, mainly occurring in the cross resistance patterns LAM + LdT and LAM + LdT + ETV, and significantly more critical in males. These results demonstrate that all people free from HBV infection should obtain injections of the vaccine or booster shots, and conventional virologic detection in a clinical laboratory center should incorporate genotype and mutation alongside the serological factors for etiology and develop better classification methods, such as sequencing.

Differential Activation of NLRP3, AIM2, and IFI16 Inflammasomes in Humans with Acute and Chronic Hepatitis B.

Nod-like receptor protein 3 (NLRP3), absent in melanoma 2 (AIM2), and interferon gamma inducible protein 16 (IFI16) are innate immune sensors for intracellular microbes, which can be activated by various dangerous signals and subsequently lead to caspase-1 (CASP1) activation and the maturation cleavage of effector molecules pro-IL-1ß and pro-IL-18. Their roles in immunopathology of acute and chronic hepatitis B virus (HBV) infection are still unclear. In this study, we first investigated the activation of NLRP3, AIM2, and IFI16 inflammasomes in peripheral blood mononuclear cells (PBMCs) from patients infected with acute hepatitis B (AHB) and chronic hepatitis B (CHB) by quantitative real-time PCR and enzyme-linked immunosorbent assay. We next analyzed the impact of hepatitis B e antigen (HBeAg) on activation of AIM2 and IFI16 inflammasomes in PBMCs of CHB patients stimulated in vitro with AIM2 and IFI16 agonist ligands, poly (dA:dT) and VACA-70mer, respectively. The results showed that the mRNA expression levels of AIM2, IFI16, and CASP1 in PBMCs from AHB and CHB patients were both upregulated. Furthermore, the mRNA levels of AIM2 and IFI16 in CHB patients were significantly positively correlated with serum HBV loads. However, only in patients with AHB there was elevation of serum IL-1ß and IL-18. There was no activation of NLRP3, AIM2, and IFI16 inflammasomes in CHB patients. Stimulation of PBMCs of CHB patients in vitro with poly (dA:dT) and VACA-70mer induced the activation of AIM2 and IFI16 inflammasomes, respectively. This ligand-induced activation was suppressed by HBeAg. Our results suggest that there exists activation of the AIM2 and IFI16 inflammasomes, but not the NLRP3 inflammasome, in AHB, and the activation of the AIM2 and IFI16 inflammasomes can be inhibited by HBeAg in CHB, which may contribute to HBV-induced immunotolerance.

[Analysis of Cut-off Value in Screening of Thalassemia by Capillary Hemoglobin Electrophoresis for Pregnant Women from Shenzhen Region of China].

OBJECTIVE: To investigate the cut-off value in screening of thalassemia in pregnant women from Shenzhen region by capillary hemoglobin electrophoresis. METHODS: The data of capillary hemoglobin electrophoresis and genetic diagnosis of thalassemia from 2122 examined prenatal women were retrospectively analyzed. Capillary hemoglobin electrophoresis and α-, ß- genetic diagnosis of thalassemia were carried out for every woman. Hemoglobin electrophoresis was performed using Capillarys 2 full-automated electrophoresis instrument. Gap polymerase chain reaction and reverse dot blot were used for genetic diagnosis of thalassemia genotyping test. The cut-off value in screening of thalassemia was determined by receiver operating characteristic curve and next to analyze the value of HbA2 and HbF in screening of thalassemia using the decided cut-off value. RESULTS: The areas under the curve (AUC(Roc)) of HbA2 for diagnosis of α-, ß- thalassemia were 0.75 and 0.981 respectively, and the AUC(Roc) of HbF for diagnosis of ß-thalassemia was 0.787. When HbA2 ≤ 2.55 was taken as the cut-off value of HbA2 for diagnosis of α-thalassemia, the sensitivity, specificity, positive likelihood ratio (LR(+)) and negative likelihood ratio (LR(-)) were 89.5%, 54.8%, 1.98, 0.19 respectively. When HbA2 ≥3.9 was taken as the cut off value of HbA2 for diagnosis of ß-thalassemia, the sensitivity, specificity, LR(+) and LR(-) were 96.1%, 99.8% 480.5, 0.04 respectively. When HbF ≥0.75 was taken as the cut off value of HbF for diagnosis of ß-thalassemia, the sensitivity, specificity, LR(+) and LR(-) were 83.6%, 61.8% respectively. CONCLUSION: The cut-off value in screening of thalassemia by capillarys 2 full automated electrophoresis instrument is different from that of the traditional method of hemoglobin electrophoresis, such as cellulose acetate membrane electrophoresis and agarose gel electrophoresis. Each laboratory should establish their own respective cut off value.

[Investigation on HPV viral load and high risk HPV types infection among patients with infertility].

OBJECTIVE: High risk human papilomavirus (HPV) infection is often related to cervical cancer. This study investigated the infection of high risk HPV in cervical epithelia among infertile patients. Relative quantification and absolute quantification were applied for determination of "real" HPV viral load in the clinical setting. METHODS: Adopting multi-channels real time PCR to genotype and quantify eight high risk HPV (HPV16, 18, 45, 31; intermediate risk types: HPV33, 52, 58, 67) DNA in cervical epithelia of the 130 infertile patients and the 150 controls. This study applied housekeeping gene (beta-globin) for the DNA quantification on secretions samples for clinical diagnosis. RESULTS: The infection rate of the infertility group was 25.38 percent (33/130) and that of the control group was 11.33 percent (17/150), the difference was statistically significant. Among the 33 positive cases in the infertility group, 24 cases showed a viral load no less than 106; in 9 of them, the viral load was less than 106. Among the 17 positive cases in the control group, 4 cases had a viral load no less than 106; in 13 of them, the viral load was less than 106. There is a statistically significant difference in viral load between the infertility group and the control group. CONCLUSION: The HPV infection rate of the infertility group was higher than that of the control group.

[Apoptotic effect of bcl-XL antisense oligodeoxynucleotide mediated by lipofectin on cell strain CNE-2Z].

BACKGROUND & OBJECTIVE: Recent studies have shown that overexpression of bcl-XL was detected in human nasopharyngeal carcinoma (NPC) cell strain CNE-2Z, suggesting it may play a pivotal role in tumorigenesis of NPC. The current study was designed to explore the effect of bcl-XL antisense oligodeoxynucleotide (ASODN) on CNE-2Z. METHODS: A 20-mer gapmer ASODN with a full phosphorothioate backbone targeting a sequence unique of the bcl-XL coding region was artificially synthesized. Bcl-XL ASODN was transfected into CNE-2Z cells through lipofectin. The survival rate was assessed by MTT assay and internucleosomal fragmentation of genomic DNA was detected by agarose gel electrophoresis. Apoptotic changes after treatment with ASODN were observed by fluorescence microscopy and flow cytometry. RESULTS: MTT assay showed that the proliferation of CNE-2Z cells decreased significantly after treatment with ASODN/Lip as compared with control (P < 0.01). ASODN/Lip reduced the proliferation of CNE-2Z in a dose-dependent manner. After treatment with ASODN/Lip for 36 hours, most cells stained with Hoechst 33258/Pl exhibited apoptotic cell morphology such as cell shrinkage, nuclear condensation, and nuclear fragmentation under fluorescence microscope; a apoptotic peak appeared on flow cytometry; a ladder-like pattern of DNA fragmentation appeared on agarose gel electrophoresis. CONCLUSION: ASODN can inhibit proliferation of CNE-2Z cells and induce apoptosis of CNE-2Z cells. The results suggest that bcl-XL is a promising target for gene therapy of NPC.