Microfluidic Streptomyces Cultivation for Whole Lifecycle Characterization and Phenotypic Assays Enabled by Nanogap-stabilized Air-Water Interface

Streptomyces is a model filamentous prokaryote to study multicellular differentiation and a rich reservoir for antibiotics discovery. In their natural conditions, Streptomyces grows at the interface of porous soil, air, and water. The morphological development of Streptomyces is traditionally performed on agar plates and mostly studied at the population levels. However, the detailed lifecycle of Streptomyces has not been well studied due to its complexity and lack of research tools which can mimic their natural conditions in the soil. Here, we developed a simple assembled microfluidic device for cultivation and the entire lifecycle observation of Streptomyces development from single-cell level. The microfluidic device composed of a microchannel for loading samples and supplying nutrients, microwell arrays for seeding and growth of single spores, and air-filled chambers aside of the microwells that facilitate growth of aerial hyphae and spores. A unique feature of this device is that each microwell is surrounded by a 1.5 μm gap connected to an air-filled chamber which provide stabilized water-air interface. We used this device to observe the development of single Streptomyces spores and found that unlike those in bulk liquid culture, Streptomyces can differentiate at water-air interfaces in microscale liquid culture. Finally, we demonstrated that phenotypic A-Factor assay can be performed at defined time point of its lifecycle. This microfluidic device could become a robust tool for studying Streptomyces multi-cellular differentiation and interaction at single cell level.

The Evaluation of APTT Reagents in Reference Plasma, Recombinant FVIII Products; Kovaltry® and Jivi® Using CWA, Including sTF/7FIX Assay

The FVIII activity in patients treated with several extended half-life FVIII (EHL-FVIII) agents different when various activated partial thromboplastin time (APTT) reagents were used. The present study examined the difference in clot waveform analysis (CWA) findings and FVIII activity when various APTT reagents and CWA were used. The CWA including FVIII activity was measured using 12 APTT reagents, and the FIX activation based on a small amount of tissue factor assay (sTF/FIX) were examined in reference plasma (RP), EHL-FVIII (Jivi®) and Kovaltry®. The 3 APTT reagents were associated with high variation in the peak time and height in the CWA when analyzing low concentrations of FVIII. The peak time and height could not be measured with one APTT reagent, and there were marked differences in the CWA findings between Jivi® and Kovaltry® among APTT reagents. Several APTT reagents showed a markedly lower FVIII activity with Jivi® than with Kovaltry®. In the FVIII assay, the peak time measured with sTF/FIX did not differ markedly between Jivi® and Kovaltry®; however, the FVIII activity in Jivi® (as measured by the peak height) tended to be higher than in Kovaltry®. The CWA findings for monitoring Jivi® varied for monitoring Jivi® depending on the APTT reagents used, and sTF/FIX assay may be able to measure the EHL-FVIII.

Inhibitor development in mild haemophilia after a major surgery for periampullary cancer (Whipple’s procedure) in an elderly man

Around the world, with the availability of factor concentrates, patients with haemophilia have undergone major and minor surgeries. Inhibitor development in early postoperative period leading to inadequate factor recovery and ongoing bleeding is a nightmare for both operating surgeon as well as haematologists. We describe a case of an elderly man with mild haemophilia A, who was diagnosed with pancreatic carcinoma and underwent Whipple’s procedure. After an uneventful procedure, he developed high-titre inhibitors and bleeding a week after surgery posing major challenges in his management. The case highlights the importance of experienced surgeons, trained haematologists, regular monitoring of factor assay/inhibitors, adequate factor and bypassing-agent support while performing such procedures.

New Hydroquinone Monoterpenoid and Cembranoid-Related Metabolites from the Soft Coral Sarcophyton tenuispiculatum

Chemical investigation of the marine soft coral Sarcophyton tenuispiculatum resulted in the isolation of a 1,4-dihydrobenzoquinone, sarcotenuhydroquinone (1), three new cembranoids, sarcotenusenes A‒C (2‒4), and ten previously reported metabolites 5–14. The chemical structures of all isolated metabolites were determined by detailed spectroscopic analyses. In biological assays, anti-inflammatory, cytotoxic, and peroxisome proliferator-activated receptor γ (PPAR-γ) transcription factor assays of all compounds were performed. None of the isolated compounds were found to exhibit activity in the PPAR-γ transcription factor assay. The anti-inflammatory assays showed that (+)-7α,8β-dihydroxydeepoxysarcophine (13) inhibited the production of IL-1β to 56 ± 1% at a concentration of 30 µM in lipopolysaccharide (LPS)-stimulated J774A.1 macrophage cells. In addition, 1 and 2 were found to exhibit cytotoxicity towards a panel of cancer cell lines.

Assessing acute and chronic Staphylococcus aureus growth and virulence in an ex vivo model of cystic fibrosis lung infection

Staphylococcus aureus is routinely found in sputum samples obtained from people with Cystic Fibrosis (CF). However, its role in the progression of the disease is unclear. This is important, as antibiotic clearance of S. aureus in CF yields unclear clinical results and there is debate around the utility of anti-Staphylococcal antibiotic treatment. We used an ex vivo porcine lung model (EVPL) to compare the growth and virulence of S. aureus isolates from acute CF exacerbations, with isolates from the same donors when they were stable. There was no significant difference in mean bacterial load between donors, strains or clinical state. However, when we compared the variance in bacterial load of each pair of exacerbation/stable isolates across experimental replicates of the lung model, we found that stable samples grew more consistently in the EVPL compared to those taken from the same donor during an exacerbation. Virulence factor assay results were mixed, with results implying greater virulence in either stable or acute samples after passage through the EVPL. We could not detect the AIP quorum sensing signal, which control expression of numerous acute virulence factors, using a reporter assay. We hypothesise that S. aureus might down-regulate Agr expression in the model, consistent with a role as a silent persister, rather than as a pathogenic agent. Further work using the EVPL model will determine how well this reflects the clinical reality in CF.

ABO blood group, glycosyltransferase activity and risk of Venous Thrombosis

IntroductionABO blood group influence the risk of venous thrombosis (VT) by modifying A and B glycosyltransferases (AGT and BGT) activities that further modulates Factor VIII (FVIII) and von Willebrand Factor (VWF) plasma levels. The aim of this work was to evaluate the association of plasma GTs activities with VWF/FVIII plasma levels and VT risk in a case-control study.Materials and Methods420 cases were matched with 420 controls for age and ABO blood group. GT activities in plasma were measured using the quantitative transfer of tritiated N-acetylgalactosamine or galactose to the 2’-fucosyl-lactose and expressed in disintegration per minute/30µL of plasma and 2 hours of reaction (dpm/30µL/2H). FVIII and VWF plasma levels were respectively measured using human FVIII-deficient plasma in a 1-stage factor assay and STA LIATEST VWF (Diagnostica Stago).ResultsA and B GT activities were significantly lower in cases than in controls (8119±4027 vs 9682±4177 dpm/30µL/2H, p=2.03 × 10−5, and 4931±2305 vs 5524±2096 dpm/30µL/2H, p=0.043 respectively). This association was observed whatever the ABO blood groups. The ABO A1 blood group was found to explain∼80% of AGT activity. After adjusting for ABO blood groups, AGT activity was not correlated to VWF/FVIII plasma levels. Conversely, there was a moderate correlation (ρ∼0.30) between BGT activity and VWF/ FVIII plasma levels in B blood group carriers.ConclusionThis work showed, for the first time, that GT activities were decreased in VT patients in comparison to controls with the same ABO blood group. The biological mechanisms responsible for this association remained to be determined.

Congenital Vitamin K-Dependent Clotting Factors Deficiency Type 1: A Rare Bleeding Disorder

Combined deficiency of vitamin K-dependent clotting factors is usually an acquired clinical problem, often resulting from liver disease, malabsorption or warfarin overdose. However, an inherited form of the disease is very rare. Here we report a 4-month-old girl who presented with a 2-week history of multiple bruises and a 1-day history of right thigh swelling after receiving her 4th month vaccine. Laboratory investigations showed anemia (Hb 6.0 g/dL) with extremely prolonged PT and APTT. Factor assay revealed deficiency of vitamin K-dependent clotting factors II, VII, IX, X as well as protein C and protein S. Whole-exome sequencing detected a novel homozygous mutation (c.44-5T>A p.(?)) in the γ-glutamyl carboxylase (GGCX) gene responsible for the autosomal recessive combined vitamin K-dependent clotting factors deficiency type 1.