Identification and characterization of the capsule depolymerase Dpo27 from phage IME-Ap7 specific to Acinetobacter pittii.

Among the Acinetobacter genus, Acinetobacter pittii stands out as an important opportunistic infection causative agent commonly found in hospital settings, which poses a serious threat to human health. Recently, the high prevalence of carbapenem-resistant A. pittii isolates has created significant therapeutic challenges for clinicians. Bacteriophages and their derived enzymes are promising therapeutic alternatives or adjuncts to antibiotics effective against multidrug-resistant bacterial infections. However, studies investigating the depolymerases specific to A. pittii strains are scarce. In this study, we identified and characterized a capsule depolymerase, Dpo27, encoded by the bacteriophage IME-Ap7, which targets A. pittii. A total of 23 clinical isolates of Acinetobacter spp. were identified as A. pittii (21.91%, 23/105), and seven A. pittii strains with various K locus (KL) types (KL14, KL32, KL38, KL111, KL163, KL207, and KL220) were used as host bacteria for phage screening. The lytic phage IME-Ap7 was isolated using A. pittii 7 (KL220) as an indicator bacterium and was observed for depolymerase activity. A putative tail fiber gene encoding a polysaccharide-degrading enzyme (Dpo27) was identified and expressed. The results of the modified single-spot assay showed that both A. pittii 7 and 1492 were sensitive to Dpo27, which was assigned the KL220 type. After incubation with Dpo27, A. pittii strain was susceptible to killing by human serum; moreover, the protein displayed no hemolytic activity against erythrocytes. Furthermore, the protein exhibited sustained activity across a wide pH range (5.0-10.0) and at temperatures between 20 and 50°C. In summary, the identified capsule depolymerase Dpo27 holds promise as an alternative treatment for combating KL220-type A. pittii infections.

Near Full-Length Genome Characterization of Two Novel Unique Recombinants (CRF01_AE/CRF07_BC) in Beijing, China.

With the prevalence of human immunodeficiency virus type 1 (HIV-1) CRF01_AE and CRF07_BC subtypes in China, the co-circulation of multiple subtypes in the HIV-1-positive population may result in dual infection or superinfection in the population, leading to the emergence of unique recombinant forms (URFs) of the HIV-1 virus. In this study, two second-generation unique recombinant strains, BI0114 and BI0116, were identified, and their near full-length genome sequences were obtained. Recombination analysis showed that both sequences were isoforms of URF_0107, and they were second-generation unique recombinant strains formed by the recombination of CRF01_AE and CRF07_BC, with the isoforms being CRF01_AE and CRF0107_BC, respectively. The continued emergence of novel CRF01_AE/CRF07_BC recombinant strains suggests that the epidemiological, preventive, and control situation of HIV-1 is complex and that the relevant health authorities urgently need to establish responses to the challenges posed by changes in the pattern of strain recombination.

Circulating mitochondrial DNA is associated with anemia in newly diagnosed hematologic malignancies.

Recent reports discovered that red blood cells (RBCs) could scavenge cell-free mitochondrial DNA (mtDNA), which drives the accelerated erythrophagocytosis and innate immune activation characterized by anemia and inflammatory cytokine production. However, the clinical value of the circulating mtDNA copy number alterations in hematologic malignancies is poorly understood. Our data showed that in comparison to healthy group, the patients group had significantly higher mtDNA and histone H4 levels. Moreover, we observed that RBC-bound mtDNA and histone H4 were negatively correlated with hemoglobin in patients. In addition, cytokines and chemokines levels in patients differed significantly from normal controls (21 higher, 7 lower). Our study suggested that both circulating mtDNA and histone H4 were associated with anemia in hematologic malignancies, which helps to further understand the potential mechanism of anemia development in patients with hematologic malignancies. This information may play a vital role in the specific therapeutic interventions for leukemia in the future.

Platelet 3D Preservation Using a Novel Biomimetic Nanofiber Peptide for Reduced Apoptosis and Easy Storage.

Human platelets (PLTs) are vulnerable to unfavorable conditions, and their adequate supply is limited by strict transportation conditions. We report here that PLTs preserved under three-dimensional (3D) conditions using novel biomimetic nanofiber peptides showed reduced apoptosis compared with classical PLTs stored at 22 °C and facilitated the storage and transportation of PLTs. The mechanism of PLT 3D preservation involves the formation of cross-links and a 3D nanofibrous network by a self-assembled peptide scaffold material at physiological conditions after initiation by triggers in plasma. PLTs adhere to the surface of the nanofibrous network to facilitate the 3D distribution of PLTs. The 3D microstructure, rheological properties, and effect on the inflammatory response and hemolysis were evaluated. Compared to traditional PLTs stored at 22 °C, PLTs subjected to 3D preservation showed similar morphology, number, aggregation activity, and reduced apoptosis. The detection of the reactive oxygen species (ROS) levels demonstrated that both reduced intracellular and mitochondrial ROS levels were correlated with reduced apoptosis. This study reveals a new 3D preservation method for PLTs based on the use of novel biomimetic nanofiber peptides that presents an attractive opportunity for various biomedical applications.

[Establishment and Application of HPA1-6, 15 Platelet Donor Bank in Beijing Area].

OBJECTIVE: To establise the bank of platelet donors with the human platelet antigen (HPA) 1-6, 15 genes so as to provide the HPA-matched platelets for the patients. METHODS: The HPA genotyping of platelets donors and patients with platelet antibody positive confirmed by sercening was performed by using the SSP-PCR; the efficacy of transfusing the HPA-matched platelets for 37 cases platelet antibody positive was analyzed. RESULTS: The most common genotype in platelet donors were HPA-1a/1a-2a/2a-3a/3b-4a/4a-5a/5a-6a/6a-15a/15b, followed by HPA-1a/1a-2a/2a-3a/3a-4a/4a-5a/5a-6a/6a-15a/15b; the most common genotype in 53 cases of platelet antibody positive confirened by screening were HPA-1a/1a-2a/2a-3a/3b-4a/4a-5a/5a-6a/6a-15a/15b. Among 37 patients with platelet antibody positive confirened by screeming, 28 showed that the transfusion of HPA-matched platelets was effective with statistically significant difference in comparison with random transfusion group. The HPA-3, HPA-15 were the main factors leading to polymorphisms. CONCLUSION: HPA-3 and HPA-15 are polymorphic, which should be focused on. HPA-matched platelets can improve the efficiency of platelet transfusion, and avoid the waste of blood resources. The genotypes of platelet donors can basically meet the requirements for common genotype transfusion.

Effect of phase-change material blood containers on the quality of red blood cells during transportation in environmentally-challenging conditions.

BACKGROUND: The effect of phase-change material blood containers on the quality of stored red blood cells (RBCs) transported in the Qinghai-Tibet Plateau remains to be studied. STUDY DESIGN AND METHODS: RBCs stored in a phase-change material blood container were transported from Chengdu to Tibet and then back to Chengdu. The detection time points were the 1st day of fresh-collected RBCs (group 1), the 14th day of resting refrigerated storage (group 2), and the 14th day of plateau transportation under refrigerated storage in the container (group 3). RBC counts, hemoglobin (HGB) content, free hemoglobin (FHb) content, blood biochemical indexes, hemorheologic indexes and 2,3-DPG content were detected. RESULTS: Compared with group 2, RBC counts and HGB were decreased, and the mean corpuscular volume (MCV), FHb and K+ content were increased in group 3. The glucose consumption and lactic acid production were significantly increased in groups 2 and 3. Compared with group 2, the 2,3-DPG content and whole blood viscosity were decreased in group 3. After resting refrigerated storage and plateau transportation, the RBC quality still met the national standard (GB18469-2012 whole blood and component blood quality requirements). CONCLUSION: The phase-change material blood container can be maintained at a constant temperature under plateau environmental conditions, ensuring that the quality of the stored RBCs is compliant with GB18469-2012 whole blood and component blood quality requirements.

Toxic effects of dimethyl sulfoxide on red blood cells, platelets, and vascular endothelial cells in vitro.

Dimethyl sulfoxide (DMSO) is widely used in biological studies as a cryoprotective agent for cells and tissues, and also for cryopreserved platelets (PLTs). However, few data on the toxic effects of DMSO following intravenous infusion of cryopreserved PLTs are available. The aim of this study was to explore dose-related effects of DMSO on red blood cells (RBCs), PLTs and vascular endothelial cells in vitro. The results showed that DMSO treatments had significant effects on RBCs, affecting osmotic fragility and increasing hemolysis. Free hemoglobin (FHb) level of RBCs was 0.64 ± 0.19 g L-1 after incubation for 6 h with 0.6% DMSO, and these levels were elevated compared with controls (0.09 ± 0.05 g L-1). Aggregation of PLTs induced by adenosine diphosphate, thrombin (THR), and thrombin receptor activator peptide (TRAP) were inhibited by DMSO treatment because the THR generation capacity was reduced. The intensity of the cytosolic esterase-induced fluorescence response from carboxy dimethyl fluorescein diacetate (CMFDA) in PLTs was decreased about 29% ± 0.04% after treatment with DMSO. DMSO also inhibited the proliferation of the vascular endothelial cell line EAhy926 cells by blocking the G1 phase. Apoptosis of EAhy926 cells with 0.6% DMSO stimulation was increased threefold compared to controls. On the basis of these findings, it was concluded that DMSO was toxic to the hematologic system. This should be taken into account when assessing the infusion effects of cryopreserved PLTs or other blood products requiring DMSO as a vehicle, such as cryopreserved stem cells, in order to avoid adverse therapeutic effects.

Evaluation of group A1B erythrocytes converted to type as group O: studies of markers of function and compatibility.

BACKGROUND: Enzymatic conversion of blood group A1B red blood cells (RBC) to group O RBC (ECO) was achieved by combined treatment with α-galactosidase and α-N-acetylgalactosaminidase. The aim of this study was to evaluate the function and safety of these A1B-ECO RBC in vitro. MATERIALS AND METHODS: A 20% packed volume of A1B RBC was treated with enzymes in 250 mM glycine buffer, pH 6.8. The efficiency of the conversion of A and B antigen was evaluated by traditional typing in test tubes, gel column agglutination technology and fluorescence-activated cell sorting (FACS) analysis. The physiological and metabolic parameters of native and ECO RBC were compared, including osmotic fragility, erythrocyte deformation index, levels of 2,3-diphosphoglycerate, ATP, methaemoglobin, free Na(+), and free K(+). The morphology of native and ECO RBC was observed by scanning electron microscopy. Residual α-galactosidase or α-N-acetylgalactosaminidase in A1B-ECO RBC was detected by double-antibody sandwich ELISA method. Manual cross-matching was applied to ensure blood compatibility. RESULTS: The RBC agglutination tests and FACS results showed that A1B RBC were efficiently converted to O RBC. Functional analysis suggested that the conversion process had little impact on the physiological and metabolic parameters of the RBC. The residual amounts of either α-galactosidase or α-N-acetylgalactosaminidase in the A1B-ECO RBC were less than 10 ng/mL of packed RBC. About 18% of group B and 55% of group O sera reacted with the A1B-ECO RBC in a sensitive gel column cross-matching test. DISCUSSION: The conversion process does not appear to affect the morphological, physiological or metabolic parameters of A1B-ECO RBC. However, the A1B-ECO RBC still reacted with some antigens. More research on group O and B sera, which may partly reflect the complexity of group A1 the safety of A1B-ECO RBC is necessary before the application of these RBC in clinical transfusion.

Label-Free Isolation and mRNA Detection of Circulating Tumor Cells from Patients with Metastatic Lung Cancer for Disease Diagnosis and Monitoring Therapeutic Efficacy.

We develop an inertial-based microfluidic cell sorter combined with an integrated membrane filter, allowing for size-based, label-free, and high-efficiency separation and enrichment of circulating tumor cells (CTCs) in whole blood. The cell sorter is composed of a double spiral microchannel that hydrodynamically focuses and separates large CTCs from small blood cells. The focused CTCs with the equilibrium position around the midline of microchannel are further captured and enriched by a membrane filter (pore size of 8 µm) attached at the middle outlet. This integrated microfluidic device can process 1 mL of whole blood containing spiked tumor cells (A549, human lung adenocarcinoma epithelial cell line) within 15 min, with the capture efficiency of 74.4% at the concentration as low as tens of A549 cells per mL of whole blood. This microfluidic cell sorter is further adopted for isolation of CTCs from peripheral blood samples of patients with metastatic lung cancer. The immunostaining and CK-19 mRNA detection are applied for identification of captured CTCs, showing that our method can detect 90% of metastatic lung cancer patients before therapy, whereas the commercially used system can only detect 40% of the same patients. We also use the expression of CK-19 mRNA from captured CTCs as an indicator for monitoring the therapeutic efficiency, which correlates well with X-ray computed tomography (CT) assessment of the disease.

[Evaluation of Storage Performance of Preserving Bags for Manually Separated Platelets].

OBJECTIVE: To evaluate the storage performance of the domestically made platelet storage bags (experimental group) and the United States Trima set platelet storage bags (control group). METHODS: The manually separated platelets were divided in two equal parts, which was added to control blood bags and experimental blood bags respectively, all samples were stored at a 22 °C ± 2 °C. The platelet count, mean volume, aggregation activity (ADP, THR), pH, glucose, lactate concentration, lactate dehydrogenase concentration, hypotonic shock reaction, CD62P and phosphatidic acid serine content were detected at day 0, 3, 5 and 7 of storage. RESULTS: There was no significant difference of platelet quality at day 5 after storage between the experimental group and the control group (T-test, P > 0.05). CONCLUSION: Two kinds of platelet storage bags have the similar storage performance.

Dysregulation of the miR-324-5p-CUEDC2 axis leads to macrophage dysfunction and is associated with colon cancer.

CUEDC2, a CUE-domain-containing protein, modulates inflammation, but its involvement in tumorigenesis is still poorly understood. Here, we report that CUEDC2 is a key regulator of macrophage function and critical for protection against colitis-associated tumorigenesis. CUEDC2 expression is dramatically upregulated during macrophage differentiation, and CUEDC2 deficiency results in excessive production of proinflammatory cytokines. The level of CUEDC2 in macrophages is modulated by miR- 324-5p. We find that Cuedc2 KO mice are more susceptible to dextran-sodium-sulfate-induced colitis, and macrophage transplantation results suggest that the increased susceptibility results from the dysfunction of macrophages lacking CUEDC2. Furthermore, we find that Cuedc2 KO mice are more prone to colitis-associated cancer. Importantly, CUEDC2 expression is almost undetectable in macrophages in human colon cancer, and this decreased CUEDC2 expression is associated with high levels of interleukin-4 and miR-324-5p. Thus, CUEDC2 plays a crucial role in modulating macrophage function and is associated with both colitis and colon tumorigenesis.

Impact on storage quality of red blood cells and platelets by ultrahigh-frequency radiofrequency identification tags.

BACKGROUND: Compared to ISBT128 code labels, radiofrequency identification (RFID) tags have incomparable advantages and gradually applied in blood management system. However, there is no global standard for the uses of RFID frequency. Even though ISBT recommended high-frequency RFID with 13.56MHz, 820- to 960-MHz ultrahigh frequency (UHF) RFID technology in many ways has even more advantages. For this reason, we studied the effect of UHF RFID tags with 820- to 960-MHz exposure on storage quality of red blood cells (RBCs) and platelets (PLTs). STUDY DESIGN AND METHODS: Thirty units of collected and prepared suspended RBCs (sRBCs) and PLTs were divided into two bags, one each for the test and control groups. The sRBCs were stored in 4±2°C refrigerator and the PLTs in a 22±2°C rocking box. The test groups were exposed to RF reader continuously during storage. Sampling at different time points and biologic changes were tested. RESULTS: As the extension of storage and the pH and chlorine levels in the supernatant of sRBCs were reduced, free hemoglobin, potassium, and sodium increased, but were not significant between test and control groups (p>0.05). During the storage period, the pH levels, PLT count, and PLT aggregation rate were decreased in both test and control groups, but were not significant (p>0.05). CONCLUSION: When exposed to 820- to 960-MHz RF, the biologic and biochemical indexes are not found to be exacerbated during 35 days of storage for sRBCs and 5 days for PLTs, respectively.

[Relationship between amount of HBV DNA in serum/liver tissue and hepatitis G virus (HGV) infection in patients with chronic hepatitis B].

OBJECTIVE: To observe the relationship between the amount of HBV DNA in serum/liver tissue and HGV infection in patients with chronic hepatitis B (CH-B) for exploring the effect of HGV infection on hepatitis B virus (HBV) replication of CH-B. METHODS: HGV RNA in serum, HGV nonstructural region 5 (NS5) antigen (HGV Ag) in liver tissue and the amount of HBV DNA in serum, liver tissue were detected for 56 patients with CH-B by reverse transcription-polymerase chain reaction (RT-PCR) assay, peroxidase antiperoxidase (PAP) immunohistochemical method and fluorescence quantitative PCR assay, respectively. Then the relationship between HGV Ag expression in liver tissue and HGV RNA expression in serum was analysed and the amount of HBV DNA in serum and liver tissues from the serum HGV RNA or liver tissue HGV Ag positive patients were compared with those of the serum HGV-RNA or liver tissue HGV Ag negative patients, respectively. RESULTS: Ten (17.9%) and eight (14.3%) patients were positive for serum and liver tissues,respectively.HGV RNA expression in serum was closely related to HGV Ag expression in liver tissues, but there was HGV RNA in serum from some of the liver tissues HGV Ag negative patients ?cases of HGV RNA and HGV Ag positive or negative,HGV RNA positive but HGV Ag negative, HGV RNA negative but HGV Ag positive, respectively: 5,43,5,3,(P<0.01). There was no significant difference in the amount of HBV DNA in serum and liver tissues between HGV RNA or HGV Ag positive and negative patients (P>0.05). CONCLUSIONS: HGV infection may not affect HBV replication. Liver is the site of HGV replication, but HGV probably also replicates in extrahepatic tissues. HGV hepatic pathogenicity is probably mild and further studies are still needed.