Blood Hemolysis Research Articles

Safety and Efficacy of a Novel Centrifugal Pump and Driving Devices of the OASSIST ECMO System: A Preclinical Evaluation in the Ovine Model.

Background: Extracorporeal membrane oxygenation (ECMO) provides cardiopulmonary support for critically ill patients. Portable ECMO devices can be applied in both in-hospital and out-of-hospital emergency conditions. We evaluated the safety and biocompatibility of a novel centrifugal pump and ECMO device of the OASSIST ECMO System (Jiangsu STMed Technologies Co., Suzhou, China) in a 168-h ovine ECMO model.Methods: The portable OASSIST ECMO system consists of the control console, the pump drive, and the disposable centrifugal pump. Ten healthy sheep were used to evaluate the OASSIST ECMO system. Five were supported on veno-venous ECMO and five on veno-arterial ECMO, each for 168 h. The systemic anticoagulation was achieved by continuous heparin infusion to maintain the activated clotting time (ACT) between 220 and 250 s. The rotary speed was set at 3,200–3,500 rpm. The ECMO configurations and ACT were recorded every 6 hours (h). The free hemoglobin (fHb), complete blood count, and coagulation action test were monitored, at the 6th h and every 24 h after the initiation of the ECMO. The dissection of the pump head and oxygenator were conducted to explore thrombosis.Results: Ten sheep successfully completed the study duration without device-related accidents. The pumps ran stably, and the ECMO flow ranged from 1.6 ± 0.1 to 2.0 ± 0.11 L/min in the V-V group, and from 1.8 ± 0.1 to 2.4 ± 0.14 L/min in the V-A group. The anticoagulation was well-performed. The ACT was maintained at 239.78 ± 36.31 s, no major bleeding or thrombosis was observed during the ECMO run or in the autopsy. 3/5 in the V-A group and 4/5 in the V-V group developed small thrombus in the bearing pedestal. No obvious thrombus formed in the oxygenator was observed. The hemolytic blood damage was not significant. The average fHb was 0.17 ± 0.12 g/L. Considering hemodilution, the hemoglobin, white blood cell, and platelets didn't reduce during the ECMO runs.Conclusions: The OASSIST ECMO system shows satisfactory safety and biocompatibility for the 168-h preclinical evaluation in the ovine model. The OASSIST ECMO system is promising to be applied in clinical conditions in the future.

Antioxidant Activities of Resveratrol Loaded Poloxamer 407: An In Vitro and In Vivo Study

Resveratrol (RES) is a bioactive molecule with potent antioxidant properties, and it constitutes an essential component of herbal medicine. This study was designed to use a nano-syntheses approach to encapsulate RES in Poloxamer 407 nanoparticles. This type of nano-construction has been employed in a variety of biological applications, both in vitro and in vivo. The contribution of this nano-construct is to increase antioxidant activity over the pure RES was investigate. 1,1-diphenyl-2-picryl hydrazyl (DPPH), and hydrogen peroxide radical scavenging assays were utilized in vitro. RES-loaded poloxamer 407 showed concentration-dependent scavenging action in the range of 20-80 µg.ml-1, with a maximum activity of 80.1% at 80 µg.ml-1. Whereas pure RES had a decrement of 61.7%. While the activity of positive control (Vit. C) was 93.2%. The magnitude of blood hemolysis examination was 3.9% at 80 µg.ml-1. That may indicate RES-loaded poloxamer 407 provide significant red blood cell protection. The pure RES did not prevent the appearance of abnormal cells (echinocytes), and that was cured by RES-loaded poloxamer 407. Finally, the safety of RES-loaded poloxamer 407 was assessed in vivo. Male mice were invested to detect the functions of their liver and kidney. A histopathological study was included as well. The findings showed that RES-loaded poloxamer 407 might have superior characters as a drug delivery system, nutritional supplements, and may be used in pharmaceutical products.

Reduced ectoparasite load, body mass and blood haemolysis in Eurasian kestrels (Falco tinnunculus) along an urban\u2013rural gradient

Urbanisation is proceeding at an alarming rate which forces wildlife to either retreat from urban areas or cope with novel stressors linked to human presence and activities. For example, urban stressors like anthropogenic noise, artificial light at night and chemical pollution can have severe impacts on the physiology of wildlife (and humans), in particular the immune system and antioxidant defences. These physiological systems are important to combat and reduce the severity of parasitic infections, which are common among wild animals. One question that then arises is whether urban-dwelling animals, whose immune and antioxidant system are already challenged by the urban stressors, are more susceptible to parasitic infections. To assess this, we studied nestlings of Eurasian kestrels (Falco tinnunculus) in Vienna, Austria, during 2015 and 2017. We measured biomarkers of innate immune function, oxidative stress and body mass index and ectoparasite infection intensity in 143 nestlings (from 56 nests) along an urban gradient. Nestlings in more urbanised areas had overall fewer ectoparasites, lower haemolysis (complement activity) and lower body mass index compared to nestlings in less urbanised areas. None of the other immune or oxidative stress markers were associated with the urban gradient. Despite some non-significant results, our data still suggest that kestrel nestlings experience some level of reduced physiological health, perhaps as a consequence of exposure to more urban stressors or altered prey availability in inner-city districts even though they had an overall lower ectoparasite burden in these heavily urbanised areas.

Анализ течения вязкой жидкости в элементах микронасоса для систем вспомогательного кровообращения

The paper presents the results of developing a micro-pump for the circulatory support system. The maximum diameter of the micropump is 6.5 mm. This allows it to be introduced into the human body through the femoral artery, which ensures its minimally invasive use. The micropump draws blood from the left ventricle of the heart and pumps it into the aorta behind the aortic valve. The numerical analysis of the spatial flow of an incompressible viscous fluid (blood) in the developed elements of the micropump allowed us to prove that its flow path corresponds to the conditions of minimal hemolysis and thrombosis during blood pumping. The developed micropump ensures uniform distribution of pressure and blood flow velocity at the outlet, which guarantees a uniform blood supply to the aorta. There are no zones of stagnation of blood and vortices throughout the flow part of the micropump, which reduces thrombosis in the micropump. In the entire volume of blood flow, even in the peripheral section of the micropump impeller, shear rates and shear stresses do not exceed critical values, which leads to minimal blood hemolysis in the developed elements of the micropump. The obtained results of 3D flow simulation in the elements of the micropump made it possible to develop design documentation for the manufacture and testing of its prototype on hydraulic and hemodynamic stands

Are Bordetella bronchiseptica Siphoviruses (Genus Vojvodinavirus) Appropriate for Phage Therapy-Bacterial Allies or Foes?

Bordetella bronchiseptica is a respiratory animal pathogen that shows growing resistance to commonly used antibiotics, which has necessitated the examination of new antimicrobials, including bacteriophages. In this study, we examined the previously isolated and partially characterized B. bronchiseptica siphoviruses of the genus Vojvodinavirus (LK3, CN1, CN2, FP1 and MW2) for their ability to inhibit bacterial growth and biofilm, and we examined other therapeutically important properties through genomic analysis and lysogeny experiments. The phages inhibited bacterial growth at a low multiplicity of infection (MOI = 0.001) of up to 85% and at MOI = 1 for >99%. Similarly, depending on the phages and MOIs, biofilm formation inhibition ranged from 65 to 95%. The removal of biofilm by the phages was less efficient but still considerably high (40–75%). Complete genomic sequencing of Bordetella phage LK3 (59,831 bp; G + C 64.01%; 79 ORFs) showed integrase and repressor protein presence, indicating phage potential to lysogenize bacteria. Lysogeny experiments confirmed the presence of phage DNA in bacterial DNA upon infection using PCR, which showed that the LK3 phage forms more or less stable lysogens depending on the bacterial host. Bacterial infection with the LK3 phage enhanced biofilm production, sheep blood hemolysis, flagellar motility, and beta-lactam resistance. The examined phages showed considerable anti-B. bronchiseptica activity, but they are inappropriate for therapy because of their temperate nature and lysogenic conversion of the host bacterium.

Rhizospheric bacteria as potential biocontrol agents against Fusarium wilt and crown and root rot diseases in tomato

The discovery of novel biocontrol agents requires the continuous scrutiny of native microorganisms to ensure that they will be useful on a regional scale. The goal of the present work was to discover novel antagonistic bacteria against Fusarium oxysporum ff. spp. lycopersici race 3 (Fol R3) and radicis-lycopersici (Forl) causing Fusarium wilt disease and Fusarium crown and root rot of tomatoes, respectively. High-throughput liquid antagonism screening of 1,875 rhizospheric bacterial strains followed by dual confrontation assays in 96-well plates was used to select bacteria exhibiting > 50% fungal growth inhibition. In a second dual confrontation assay in 10-cm Petri dishes, bacteria showing > 20% Fol R3 or Forl growth inhibition were further screened using a blood hemolysis test. After discarding β-hemolytic bacteria, a seedling antagonistic assay was performed to select five potential antagonists. A phylogenetic analysis of 16S rRNA identified one strain as Acinetobacter calcoaceticus (AcDB3) and four strains as members of the genus Bacillus (B. amyloliquefaciens BaMA26, Bacillus siamensis BsiDA2, B. subtilis BsTA16 and B. thuringiensis BtMB9). Greenhouse assays demonstrated that BsTA16 and AcDB3 were the most promising antagonists against Fol R3 and Forl, respectively. Pathogen biocontrol and growth promotion mechanisms used by these bacteria include the production of siderophores, biofilm, proteases, endoglucanases and indole acetic acid, and phosphate solubilization. These five bacteria exerted differential responses on pathogen control depending on the tomato hybrid, and on the growth stage of tomatoes. We report for the first time the use of an Acinetobacter calcoaceticus isolate (AcDB3) to control Forl in tomato under greenhouse conditions.

Preparation, Characterization, and In Vitro/In Vivo Evaluation of 3-O-β-D-Galactosylated Resveratrol-Loaded Polydopamine Nanoparticles.

3-O-β-D-galactosylated resveratrol (Gal-Res) was synthesized from resveratrol (Res) and 3-O-β-D-galactose (Gal) in our previous study. In order to improve the pH sensitivity and bioavailability of Gal-Res, Gal-Res nanoparticles (Gal-Res NPs) were prepared using polydopamine (PDA) as a drug carrier. The drug loading (DL %) and entrapment efficiency (EE %) of Gal-Res NPs were 46.80% and 88.06%. The average particle size, polydispersity index (PDI), and Zeta potential of Gal-Res NPs were 179.38 ± 2.83 nm, 0.129 ± 0.013, and - 28.05 ± 0.36 mV, respectively. The transmission electron microscope (TEM) showed that Gal-Res NPs had uniform spherical morphology. Compared with the fast release of raw Gal-Res, the in vitro release of Gal-Res NPs was slow and pH-sensitive. The results of the blood vessel irritation and hemolysis test demonstrated that Gal-Res NPs had good hemocompatibility. The pharmacokinetics study in rats showed that area under the curve of plasma drug concentration time (AUC0→600) and half-life (t1/2) of Gal-Res NPs were enhanced 1.82-fold and 2.19-fold higher than those of raw Gal-Res. The in vivo biodistribution results showed that Gal-Res NPs were more distributed in liver tissue than Gal-Res. Gal-Res NPs with high bioavailability and liver accumulation were hopeful drug delivery systems (DDS) to treat liver diseases.

Histological and histochemical changes in the peripheral organs of the immune system of dogs in cases of isoniazid poisoning

Most publications on isoniazid poisoning in dogs are devoted to clinical diagnostics, treatment, and prevention of the disease. Histological and histochemical changes are not fully described, though they are important in assessing the toxic effects of isoniazid. Isoniazid is used to treat tuberculosis in humans. Dogs are hypersensitive to this drug. The article highlights the results of macroscopic, histological, and histochemical studies of the dogs’ lymph nodes and spleen in cases of isoniazid poisoning. A pathological examination of 19 corpses of dogs of different ages was performed, during which isoniazid poisoning was posthumously diagnosed, based on anamnesis, clinical signs, pathological autopsy, histological, and histochemical examination. Samples of lymph nodes and spleen were fixed in a 10% aqueous neutral formalin solution, Carnoy’s solution, and Bouin’s fixative. Histoсuts were prepared using a sled microtome and stained with hematoxylin and eosin. Staining was also performed according to the techniques suggested by McManus, Brachet, and Perls. The pathomorphological changes in lymph nodes and spleen were characterized by disorganization of vascular walls and connective tissue fibers of the stroma, dilatation of veins, their overflow with hemolyzed blood, and, in cases of the long clinical course, thrombosis of small vessels. Intravascular hemolysis of erythrocytes resulted in an excessive formation of hemosiderin. Histochemically, the spleen and lymph nodes showed a significant increase in the number of hemosiderophages in the spleen’s red and white pulp and the lymph nodes’ central sinuses and pulp cords. In the spleen, mucoid swelling and necrobiotic changes in the wall structures of the arterioles and arteries progressed with a narrowing of their lumen in dogs suffering from the long clinical course. Increased permeability of the microcirculatory tract vessels of the spleen and lymph nodes, transudate formation, and the destructive changes in the reticular skeleton accompanied hemodynamic violations. A sharp change in blood rheology caused the violation of trophism and metabolism in the immune system. Lymphoid elements of the lymph nodes and white pulp of the spleen were in a state of karyorrhexis and karyolysis. The morphological study of the immune system’s peripheral organs suggests that dogs poisoned by isoniazid demonstrate hemodynamic disorders, changes in the physicochemical properties of blood (hemolysis of erythrocytes and thrombosis). This is the basis of trophic disorders, metabolic malfunctions, and the development of dystrophic processes in all structural elements of the spleen and lymph nodes.

G6Pd deficiency with severe hemolytic anemia: a case report

A 3-year-old boy presented to our atoll hospital in H.A Alif Dhidhoo, with severe pallor, jaundice, easy fatigability and recurrent episodes of passage of dark-colored urine for past 3 days. He was born mature at 39 weeks of gestation with no past significant medical history. Recent history revealed the consumption of 2 cans of fava beans and application of some medicinal herbs. On admission, physical examination revealed fever of 101 degree Fahrenheit, severe pallor, jaundice, cervical lymphadenopathy and mild hepatomegaly. Laboratory investigation results showed a hemoglobin level of 5.4 g/dl with a hemolytic blood picture and serum Bilirubin of 6mg/dl. The patient's G6PD level was measured which showed marked deficiency. Other causes of hemolytic anemia were excluded. Patient required urgent packed RBC transfusion and antibiotics for infection. He responded well to the treatment and was discharged in a stable condition. Parents were appropriately advised on the condition and the importance of avoiding certain foods and medication. Folic acid was prescribed for maintaining normal hemoglobin concentration. This is a first case report in North Maldives of G6PD presenting with severe hemolytic anemia requiring blood transfusion.

Synthesis and ex vivo evaluation of PLGA chitosan surface modulated double walled transdermal Pluronic nanogel for the controlled delivery of Temozolomide

A surface modulated biodegradable transdermal strategy has been exploited for improving the biopharmaceutical properties of Temozolomide augmented in Poly Lactic-co-glycolic acid (PLGA) chitosan double walled nanogel (PCNGL). The PCNGL was synthesized by dual approach methodology showing consistent nanosize particle range of 210 nm and PDI 0.325 ± 0.43 with cationic zeta potential values +29.34 ± 0.79 mV. The PCNGL showed qualitative endothermic & exothermic temperature dependent degradation peaks by thermogravimetry analysis. Blood hemolysis and coagulation assay showed 3.37 ± 0.19 as hemolytic ratio, validating biologically safe margin for transdermal delivery. The in vitro drug release showed 85% transdermal release at slightly acidic pH mimicking skin microenvironment. The ex vivo studies displayed noteworthy penetration potential validated by concentration depth assay and confocal laser scanning microscopy, exhibiting 80% Temozolomide uptake in porcine epidermal tissue. The current research demonstrated the biodegradable controlled delivery of chemotherapeutic Temozolomide leading to biologically safe transdermal therapy.

Screening algorithms for HBV, HCV, HIV and syphilis in an anatomical donation program

BackgroundUsers of anatomical donors are at risk of exposure to bloodborne pathogens. This study evaluated screening algorithms for hepatitis B and C virus, human immunodeficiency virus and Treponema pallidum during donor allocation and assessed the impact of postmortem time on hemolysis and how hemolysis affects test results and donor discard rate. MethodsFrom 2011 to 2018, demographic data of anatomical donors, time of postmortem blood sampling, presence of sample hemolysis, serological test results (negative; active infection; false reactive screening test; historic infection; inconclusive; technically impracticable) and the actual donor allocation were collected. ResultsDonors (n = 537) had a mean age of 77.53 ± 13.67 (24–103) year. Nine (1.68%) had laboratory test results indicative for active infection for hepatitis B (n = 1) and C virus (n = 2), human immunodeficiency virus (n = 5) and T. pallidum (n = 1). Negative screenings ranged from 74.67 to 97.58%, depending on the pathogen. According to the original screening algorithms, 479 (89.20%) donors should have been accepted. In practice, a donor acceptance rate of 91.20% was found. Analysis of potential donor allocation interpretation obstacles resulted in simplification of the in-house laboratory testing algorithms and addition of a nucleic acid test to increase the reliability for identification of active (acute) human immunodeficiency virus infection. Hemolysis was more common when sampling was performed more than 24 h after death (p < 0.001). Hemolytic samples more frequently showed a reactive or indeterminate human immunodeficiency virus test result (p < 0.001). Screening for human immunodeficiency virus and T. pallidum was technically more impracticable when hemolysis was present (p = 0.042 and p = 0.003, respectively). Donors with hemolytic blood samples were more often discarded (46.88%) compared to bodies with non-hemolytic samples (6.32%) (p < 0.001). ConclusionsDespite the implementation of donor screening algorithms, a significant number of bodies have an inconsistent allocation. New algorithms, to be evaluated in future research, were suggested. Early postmortem blood sampling is key as hemolysis can influence certain test results and donor allocation.

Synthesis, characterization and drug delivery application of Dapsone based double tailed biocompatible nonionic surfactant

The increase in antimicrobial resistance has created a crisis that has become top priority for global policy and public health. Antibiotics are constantly being rendered in-effective due to the emergence of bacterial resistance; therefore, novel strategies for improving therapeutic efficacies of existing drugs must be focused. Advancements in nanotechnology have opened up new avenues for enhancing therapeutic efficacy of existing drugs via construction of intelligent and efficient delivery systems. This study reports the synthesis of Dapsone based nonionic surfactant and its utilization as delivery system for Ceftriaxone sodium. The synthesized nonionic surfactant was characterized via mass spectrometry and 1H NMR and IR spectroscopic techniques. The drug loaded vesicles of newly synthesized sulfur based nonionic were formed through thin film hydration method and characterized for drug entrapment efficiency, vesicles size, zeta potential, morphology using UV–vis spectrometry, dynamic light scattering (DLS) and atomic force microscopic (AFM) techniques. The biocompatibility of newly synthesized surfactant was assessed using blood hemolysis and in-vitro cells cytotoxicity. Antibacterial potential of drug loaded vesicles was assessed in gram positive and gram negative bacterial cultures. The spectroscopic results confirm successful synthesis of novel sulfur based nonionic surfactant that formed spherical shaped drug loaded vesicles with an average size of 97.95 ± 3.45 nm and 56.3 ± 3.15 % entrapment of the model drug (Ceftriaxone sodium). The vesicles displayed negative surface charge of -16.8 ± 3.72 mV and released the entrapped drug in a controlled way in-vitro drug release. The drug loaded vesicular formulation showed enhanced cellular uptake and greater antibacterial potentials when compared with control. Results of this study show that the Dapsone based surfactant is safe, biocompatible, non-toxic and can be used as promising vesicular carrier for enhancing therapeutic efficacy of antibacterial drug, Ceftriaxone sodium.

Can RH+ whole blood be safely used as an alternative to RH- product? An analysis of efforts to improve the sustainability of a hospital's low titer group O whole blood program.

Low-titer group O whole blood (LTO-WB) has recently gained popularity in trauma centers for the acute resuscitation of hemorrhagic shock. However, limited supplies of Rh- product prevent implementation and strain sustainability at many trauma centers. We set out to identify whether Rh+ LTO-WB could be safely substituted for RH- product, regardless of patient's Rh status. Following Institutional Review Board approval, information on all trauma patients receiving prehospital or emergency department transfusion of uncrossed, emergency release LTO-WB (11/17-10/19) were evaluated. Patients were first divided into those who received Rh- versus Rh+ product, the assessed by Rh of the recipient. Serial hemolysis panels, transfusion reactions, and outcomes were compared. Six hundred thirty-seven consecutive trauma patients received emergency release LTO-WB. Of these, 448 received Rh+ product, while 189 received Rh- LTO-WB. Patients receiving Rh+ product were more likely to be men (81 vs. 70%) and have lower field blood pressure (median 99 vs. 109) and GCS (median 7 vs. 12); all p < 0.05. There were no differences in blood product volume, hemolysis laboratories, transfusion reactions, complications, or survival. We then separated patients by Rh status (577 were Rh+, 70 were Rh-). Rh- patients were older (median age 54 vs. 39), more likely to be women (57 vs. 26%), and more likely to have sustained blunt trauma than their Rh+ counterparts (92 vs. 70%); all p < 0.05. There were no differences in hemolysis laboratories, transfusion reactions, complications, or survival between Rh+ and Rh- patients, regardless of Rh product received. When Rh- whole blood is unavailable or in short supply, Rh+ LTO-WB appears to be a safe alternative for the resuscitation of hemorrhagic shock in both Rh+ and Rh- patients. Use of Rh+ product may help trauma centers incorporate LTO-WB into their hospital and improve sustainability of such programs. Therapeutic, Level III.

Novel molecular diagnosis of Cyl E, Spb 1 and bib A virulence genes of Streptococcus agalactiae from pregnant women

Group B Streptococcus is a gram-positive coccal bacterium which produces an arrow zone of blood hemolysis on blood agar. Group B Streptococcus is the most common cause of invasive bacterial illness in neonates, including sepsis, meningitis, septicemia, and pneumonia. For the first time, such a study of Streptococcus agalactiae has been conducted in hospitals of Anbar Governorate .All Streptococcus agalactiae identified by conventianal methods , automated methods using Vitek-2 system , and molecular methods by conventional PCR. All isolates gave a positive result as 16S rRNA. Distribution of resistance ratio for Streptococcus agalactiae were Penicillin 20% , Clindamycin 68% , Chloramphenicol % 46 , Tetracycline 82% , Erythromycin % 100 ,Imipenem % 12 , and Ciprofloxacillin 26.47%. The Cyl E and bibA genes were detected by conventional PCR and the result showed (72%) strains positive, but in case of bla Cyl E gene (53.48%) of strains harbored bla bibA genes , while 48.83 of Streptococcus agalactiae contain Spb1 gene . This approach showed the coexistence of bla Cyl E , Spb 1 and bibA genes in 48.83% strains of Streptococcus agalactiae.

Detection of Gene Mutations in Patients with Clinically Suspected Heritary Spherocytosis By Next Generation Sequencing

Introduction: Hereditary spherocytosis (HS) is the most frequent form of inherited hemolytic anemia characterized by the presence of spherical-shaped erythrocytes on the peripheral blood smear, hemolysis, splenomegaly, jaundice, and gallstones. The clinical severity of HS varied widely, and its prevalence is 1 in 2,000 in Northern European population and1 in 100,000 in China. The diagnosis can be based on the physical examination, complete red blood cell count, reticulocytes count, medical history and specific tests, preferentially the EMA test test and AGLT, as well as molecular diagnosis particularly gene sequencing. To date, mutations in at least five genes (ANK1, EPB42, SLC4A1, SPTA1, and SPTB) are associated with different subtypes of HS. Next-generation gene sequencing (NGS) is a high-throughput parallel sequencing method and its high-sensitivity is widely used in variation identification in clinical settings with a wide range of human diseases including inherited disorders. This study is aim to discover pathogenic mutations in clinically suspected HS patients by NGS to confirm and/or discriminate diagnosis.Methods: A cohort of seven patients with clinically suspected HS were enrolled in this study. The DNA samples were extracted from peripheral blood samples of the patients and their parents (if available), and were sheared into 200-300bp fragments using the Covaris ultrasonic instrument, followed by whole-exome sequencing. NGS samples were sequenced on the Illumina HiSeq X10 instrument according to manufacturer's protocol. Raw data analysis was performed with the Illumina Pipeline software and the identified mutations were confirmed by Sanger sequencing. Sequences were aligned to the hg19 human genome reference using information from databases including NCBI dbSNP, HapMap, and the 1000 Human Genomes Project dataset. SOAPsnp and Samtools were used to locate the mutations, and the potential functional impacts of the identified variants were assessed using SIFT, PolyPhen-2 and MutationTaster software.Results: Seven patients with age from 8-44yrs were enrolled in our cohort study. NGS results have identified 4 missense mutation SLC4A1 (P854L), SLC4A1 (P455R), SEC23B (P25H and P567R), 3 nonsense mutation ANK1 (R1334X), SPTB (R1756X), SPTB (E1602X), and 1 splicing mutation SPTB (c.5937+1G>C) in seven patients respectively. Three novel mutations, SPTB (R1756X and E1602X) that were stop-gain single nucleotide variants (SNVs) and splicing variant SPTB (c.5937+1G>C) were identified responsible for hereditaryspherocytosis (HS). Considering that the missense mutation SLC4A1 (P854L) was inherited from asymptomatic mother, the pathogenicity of this site is low. Moreover, the mutation SPTB (E1602X) is a de novo mutation and thus likely to be pathogenic. However, the compound heterozygous mutation SEC23B (P25H and P567R) is responsible for congenital dyserythropoietic anemia (CDA) which represent similar clinical symptoms with HS, may have been misdiagnosed and needs to be further studied.Conclusions: Together, we have identified three novel mutations, two stop-gain mutation SPTB (R1756X and E1602X) and one splicing variant SPTB (c.5937+1G>C), in hereditary spherocytosis patients. Our results suggested that genetic diagnosis by NGS is a fast, economic and accurate way to detect and identify pathogenic alterations of inherited diseases like hereditary spherocytosis, and more importantly a useful tool for differential diagnosis. DisclosuresNo relevant conflicts of interest to declare.

Effect of Paclitaxel/etoposide co-loaded polymeric nanoparticles on tumor size and survival rate in a rat model of glioblastoma

The aim of this work is to co-load paclitaxel (PTX) and etoposide (ETP) in methoxy poly(ethylene glycol)-poly(lactic-co-glycolic acid) nanoparticles (mPEG-PLGA NPs) to overcome pharmacokinetics and physiological limitations and enhance therapeutic efficacy for treating intracranial glioblastoma. Both drugs were loaded into mPEG-PLGA NPs by a nano-precipitation method. The resultant NPs demonstrated an enhanced cytotoxic effect indicated by lower IC50 values and augmented cell apoptosis to U87 and C6 glioma cell lines compared to both free drugs. Additionally, blood compatibility assays showed that the PTX/ETP co-loaded mPEG-PLGA NPs did not induce blood hemolysis, blood clotting, or platelet aggregation. In vivo anti-glioma efficacy evaluation in rats bearingintracranialC6glioma revealed a superior anti-glioma activity for the treatment with PTX/ETP co-loaded mPEG-PLGA NPs compared to other formulations, particularly a significantly longer median survival, 76 days compared to 36 days for free PTX and 37 days for free ETP treatment, respectively, and higher tumor regression, proved by magnetic resonance imaging (MRI).

Differential impacts of hemolysis on coagulation parameters of blood samples: A STROBE-compliant article.

This study aimed at investigating the impact of hemolysis on different coagulation parameters.A total of 216 venous blood samples without visible hemolysis were collected from adult patients at a tertiary referral center over six months. The plasma obtained was quantified for six coagulation parameters including prothrombin time, activated partial thromboplastin time, fibrinogen, D-dimer, antithrombin III, and protein C. The rest of the plasma from each blood sample was aliquoted into three tubes, each containing 1 mL of plasma with three different volumes of cell-free hemoglobin (i.e., 2, 4, 8 μL) from lysed RBCs to create simulated hemolyzed blood samples with hemoglobin concentration of approximately 0.1, 0.2, and 0.4 g/dL to mimic mild (1+), moderate (2+), and severe (3+) hemolysis, respectively, before repeating the coagulation tests to determine possible correlation between the simulated degree of hemolysis and the changes in test results of the coagulation parameters.Spearman correlation analysis showed significant decreases in the values of activated partial thromboplastin time, fibrinogen, D-dimer, and protein C values with an increasing degree of simulated hemolysis (all P < .01). Comparison of the percentage bias of biological variance showed significant positive associations of cell-free hemoglobin concentrations with the percentage bias of D-dimer and protein C. However, only the former was still within the range of biological variance under condition of simulated hemolysis. Besides, the presence of cell-free hemoglobin regardless of concentration had a notable impact on the percentage bias of activated partial thromboplastin time, whereas the influence was non-significant for prothrombin time, fibrinogen, and antithrombin III.The results showed different impacts of simulated hemolysis on six coagulation parameters, highlighting the dependence of clinical reliability on the coagulation parameter to be investigated in hemolytic blood samples.

Biocatalysts from cyanobacterial hapalindole pathway afford antivirulent isonitriles against MRSA

The emergence of resistance to frontline antibiotics has called for novel strategies to combat serious pathogenic infections. Methicillin-resistant Staphylococcus aureus [MRSA] is one such pathogen. As opposed to traditional antibiotics, bacteriostatic anti-virulent agents disarm MRSA, without exerting pressure, that cause resistance. Herein, we employed a thermophilic Thermotoga maritima tryptophan synthase (TmTrpB1) enzyme followed by an isonitrile synthase and Fe(II)-α-ketoglutarate-dependent oxygenase, in sequence as biocatalysts to produce antivirulent indole vinyl isonitriles. We report on conversion of simple derivatives of indoles to their C3-vinyl isonitriles, as the enzymes employed here demonstrated broader substrate tolerance. In toto, eight distinct L-Tryptophan derived α-amino acids (7) were converted to their bioactive vinyl isonitriles (3) by action of an isonitrile synthase (WelI1) and an Fe(II)-α-ketoglutarate-dependent oxygenase (WelI3) yielding structural variants possessing antivirulence against MRSA. These indole vinyl isonitriles at 10 μg/mL are effective as antivirulent compounds against MRSA, as evidenced through analysis of rabbit blood hemolysis assay. Based on a homology modelling exercise, of enzyme-substrate complexes, we deduced potential three dimensional alignments of active sites and glean mechanistic insights into the substrate tolerance of the Fe(II)-α-ketoglutarate-dependent oxygenase.

The impact of blood sampling technique, including the use of peripheral intravenous cannula, on haemolysis rates: A cohort study.

To explore the relationship between blood sampling techniques and haemolysis. Haemolysis rates of blood samples have been thought to be influenced by the method of collection. There is a lack of research evidence available to clearly show the comparative risk of haemolysis across different blood sampling methods, including venepuncture and use of peripheral intravenous cannulas. A prospective cohort study. Reporting followed the STROBE checklist. A trained observer was used to record blood sampling techniques over a 10-week period between April and June 2019. These records were then linked to pathology haemolysis results. Multivariable logistic regression was used to model patient and blood draw characteristics affecting haemolysis. Most of the blood samples were not haemolysed (n=324, 87.1%). Multivariable analysis showed haemolysis was associated with increased tourniquet duration and if the level of tube was less than half full. Univariable analysis showed haemolysis was associated with increased age of the patient, the difficulty of cannulation/ venepuncture and increased number of attempts. No difference was found in the haemolysis rate related to the qualification of the blood collector. There was no significant difference in haemolysis rates associated with sampling blood from a PIVC compared with venepuncture. Research should be undertaken to determine whether education on the factors influencing haemolysis is useful in decreasing haemolysis rates. There was no association with increased haemolysis rates when drawing blood via venepuncture compared with a peripheral intravenous cannula. Haemolysis of blood samples was associated with increased tourniquet duration, if level of the tube was less than half-filled, increased age of the patient and difficulty of blood draw. Awareness of the risk of haemolysis associated with specific blood sampling methods may assist clinicians to improve care.

Hydrocarbon solubilisation by oil and cellulose-degrading Chitinophaga terrae isolated from the rumen.

This study investigated the capacity of cellulose and hydrocarbon degrading bacterium isolated from the rumen of a cow to solubilise hydrocarbon. The bacterium was isolated from the rumen fluid of cow and its capacity to degrade cellulose was screened on carboxyl methyl cellulose (CMC) agar plate and the ability to degrade crude oil was carried out using Bonny Light crude. Solubilisation of hydrocarbon was determined by carrying out emulsification index (E24) using kerosene. Other bio-surfactant characteristics such as blood haemolysis, tilted slide capacity and oil displacement were tested also. The bacterium was identified based on phenotypic, biochemical and molecular characteristics. The isolate achieved 48.17% degradation of total petroleum hydrocarbon (TPH) within 14 days with emulsification index of 54.5%. The isolate also produced clear zone on agar plate containing CMC as the sole carbon source. Phylogenetic tree analyses classified the bacterial isolate as Chitinophaga terrae. The sequences have been deposited to GenBank under the accession number KJ076216.1. This study has demonstrated that the novel strain of Chitinophaga terrae used in this study not only has the capacity for multiple substrate utilization, but also has the capacity to produce bio-surfactant. Considering that the isolate was obtain from the rumen of cow it shows that rumen content may harbour bacteria with diverse economical and ecologically-friendly product, which may be utilized for bioremediation of crude oil contaminated systems.