Isolation, Identification, and Biological Characterization of Phage vB_KpnM_KpVB3 Targeting Carbapenem-Resistant Klebsiella pneumoniae ST11.

OBJECTIVES: This study aimed to isolate a phage capable of lysing carbapenem-resistant Klebsiella pneumoniae (CRKP) and to analyse its biological characteristics and whole-genome sequence. METHODS: The phage was isolated and purified from the sewage. Transmission electron microscopy (TEM) was employed to observe the bacteriophage's morphology. Phenotypic characterization of the bacteriophages was determined. The genomic information was analysed. Evolutionary relationships were established through comparative genomics, proteomics, and phylogenetic analysis. RESULTS: The isolation of a virulent phage, named Klebsiella phage vB_KpnM_KpVB3, was notable for forming 6-7 mm transparent circular zones, each surrounded by a distinct halo. The phage had a head diameter of ca. 30 nm and a tail length of ca. 20 nm, being identified as a member of the Myoviridae family and the Caudovirales order. The optimal multiplicity of infection (MOI) was 0.00001, with an incubation period of 20 minutes and a lysis period of 60 minutes, and the number of released phages after lysis was 133±35 PFU/cell. The phage was relatively stable at temperatures ranging from 10°C to 40°C and at pH values ranging from 3 to 11. Its lytic efficiency against CRKP was 30.30%. It has been shown to be able to destroy the biofilm of host bacteria. The bacteriophage genome consists of double-stranded DNA (dsDNA) with a total length of 48,394 base pairs, a GC content of 48.99%, and 78 open reading frames (ORFs). CONCLUSION: The study resulted in the isolation vB_KpnM_KpVB3, a phage demonstrating potential therapeutic efficacy against infections caused by CRKP.

Silk Fibroin-Based Coatings for Pancreatin-Dependent Drug Delivery.

Triggerable coatings, such as pH-responsive polymethacrylate copolymers, can be used to protect the active pharmaceutical ingredients contained within oral solid dosage forms from the acidic gastric environment and to facilitate drug delivery directly to the intestine. However, gastrointestinal pH can be highly variable, which can reduce delivery efficiency when using pH-responsive drug delivery technologies. We hypothesized that biomaterials susceptible to proteolysis could be used in combination with other triggerable polymers to develop novel enteric coatings. Bioinformatic analysis suggested that silk fibroin is selectively degradable by enzymes in the small intestine, including chymotrypsin, but resilient to gastric pepsin. Based on the analysis, we developed a silk fibroin-polymethacrylate copolymer coating for oral dosage forms. In vitro and in vivo studies demonstrated that capsules coated with this novel silk fibroin formulation enable pancreatin-dependent drug release. We believe that this novel formulation and extensions thereof have the potential to produce more effective and personalized oral drug delivery systems for vulnerable populations including patients that have impaired and highly variable intestinal physiology.

An Automation Workflow for High-Throughput Manufacturing and Analysis of Scaffold-Supported 3D Tissue Arrays.

Patient-derived organoids have emerged as a useful tool to model tumour heterogeneity. Scaling these complex culture models while enabling stratified analysis of different cellular sub-populations, however, remains a challenge. One strategy to enable higher throughput organoid cultures is the scaffold-supported platform for organoid-based tissues (SPOT). SPOT allows the generation of flat, thin, and dimensionally-defined microtissues in both 96- and 384-well plate footprints that are compatible with longitudinal image-based readouts. SPOT is currently manufactured manually, however, limiting scalability. In this study, an automation approach to engineer tumour-mimetic 3D microtissues in SPOT using a liquid handler is optimized and comparable within- and between-sample variation to standard manual manufacturing is shown. Further, a liquid handler-supported cell extraction protocol to support single-cell-based end-point analysis using high-throughput flow cytometry and multiplexed cytometry by time of flight is developed. As a proof-of-value demonstration, 3D complex tissues containing different proportions of tumour and stromal cells are generated to probe the reciprocal impact of co-culture. It is also demonstrated that primary patient-derived organoids can be incorporated into the pipeline to capture patient-level tumour heterogeneity. It is envisioned that this automated 96/384-SPOT workflow will provide opportunities for future applications in high-throughput screening for novel personalized therapeutic targets.

An off-the-shelf multi-well scaffold-supported platform for tumour organoid-based tissues.

Complex 3D bioengineered tumour models provide the opportunity to better capture the heterogeneity of patient tumours. Patient-derived organoids are emerging as a useful tool to study tumour heterogeneity and variation in patient responses. Organoid cultures typically require a 3D microenvironment that can be manufactured easily to facilitate screening. Here we set out to create a high-throughput, "off-the-shelf" platform which permits the generation of organoid-containing engineered microtissues for standard phenotypic bioassays and image-based readings. To achieve this, we developed the Scaffold-supported Platform for Organoid-based Tissues (SPOT) platform. SPOT is a 3D gel-embedded in vitro platform that can be produced in a 96- or 384-well plate format and enables the generation of flat, thin, and dimensionally-defined microgels. SPOT has high potential for adoption due to its reproducible manufacturing methodology, compatibility with existing instrumentation, and reduced within-sample and between-sample variation, which can pose challenges to both data analysis and interpretation. Using SPOT, we generate cultures from patient derived pancreatic ductal adenocarcinoma organoids and assess the cellular response to standard-of-care chemotherapeutic compounds, demonstrating our platform's usability for drug screening. We envision 96/384-SPOT will provide a useful tool to assess drug sensitivity of patient-derived organoids and easily integrate into the drug discovery pipeline.

Computationally guided high-throughput design of self-assembling drug nanoparticles.

Nanoformulations of therapeutic drugs are transforming our ability to effectively deliver and treat a myriad of conditions. Often, however, they are complex to produce and exhibit low drug loading, except for nanoparticles formed via co-assembly of drugs and small molecular dyes, which display drug-loading capacities of up to 95%. There is currently no understanding of which of the millions of small-molecule combinations can result in the formation of these nanoparticles. Here we report the integration of machine learning with high-throughput experimentation to enable the rapid and large-scale identification of such nanoformulations. We identified 100 self-assembling drug nanoparticles from 2.1 million pairings, each including one of 788 candidate drugs and one of 2,686 approved excipients. We further characterized two nanoparticles, sorafenib-glycyrrhizin and terbinafine-taurocholic acid both ex vivo and in vivo. We anticipate that our platform can accelerate the development of safer and more efficacious nanoformulations with high drug-loading capacities for a wide range of therapeutics.

[Curative effect of interleukin 11 on chronic idiopathic thrombocytopenic purpura].

The aim of this study was to investigate the circulating levels of IL-11 in the patients with chronic idiopathic thrombocytopenic purpura (CITP), and its significance, and to evaluate the curative effect of rhIL-11 on CITP. The level of IL-11 in patients with CITP was determined by ELISA before and after treatment, respectively. 1.5 mg of rhIL-11 were injected subcutaneously, once a day, continuously for 14 days as one course, treatment time 1 - 2 courses as total. The results showed that the higher blood IL-11 level was found in CITP patients than that in controls (P < 0.01) and during the course of treatment the number of platelets in peripheral blood of patients with CITP parallelled to the level of IL-11. The platelet counts were obviously increased in all CITP patients after rhIL-11 treatment. It is concluded that the serum level of IL-11 in patients is correlated to the number of platelets in patients. rhIL-11 can be used as an effective treatment for CITP.