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Background and Aims: One of the most skilled procedures performed by embryologists is intracytoplasmic sperm injection (ICSI), which takes significant experience to master. Conducting the procedure requires a high level of concentration with training a significant burden on clinical laboratory efficiency. Advancement of a micro 3D printed device to hold oocytes has removed the requirement for a holding pipette, reducing the complexity of the ICSI procedure. This study shows that the microICSIÔ device reduces the technical difficulty of ICSI, improving the speed and precision of the procedure. Method: ICSI was performed conventionally (C-ICSI) and within the microICSIÔ device on abattoir derived porcine denuded oocytes matured in vitro for 39-44 h in NCSU-23 medium containing eCG, hCG, porcine follicular fluid, EGF and insulin. Timing data was captured by video recording for pipette setup and injection time per oocyte for two embryologists and compared between each technique. Oocyte injection time was taken from when a sperm loaded injection pipette was in focus with the oocyte until the injection pipette was removed from the oocyte. Results: Results were compared between C-ICSI and microICSIÔ (Mean ± SD). Pipette setup time was reduced from 278 ± 21 seconds to 109 ± 3 seconds by removing the holding pipette. During the procedure, absence of a holding pipette resulted in embryologists adjusting their hands on average only 3 times during oocyte injection compared to 10 times for C-ICSI when moving between microinjector, pipette and microscope controllers. Oocyte traceability was also improved due to the microICSIÔ device’s numbered array. Embryologist’s injection time was reduced from 42 ± 12 seconds for C-ICSI to 14 ± 5 seconds for microICSIÔ per oocyte. Conclusion: The microICSIÔ device reduces pipette set-up time and the complexity of the ICSI procedure, reducing the time required for oocyte injection by half.

ABSTRACT The malaria parasite Plasmodium falciparum traffics the virulence protein P. falciparum erythrocyte membrane protein 1 (PfEMP1) to the surface of infected red blood cells (RBCs) via membranous organelles, known as the Maurer’s clefts. We developed a method for efficient enrichment of Maurer’s clefts and profiled the protein composition of this trafficking organelle. We identified 13 previously uncharacterized or poorly characterized Maurer’s cleft proteins. We generated transfectants expressing green fluorescent protein (GFP) fusions of 7 proteins and confirmed their Maurer’s cleft location. Using co-immunoprecipitation and mass spectrometry, we generated an interaction map of proteins at the Maurer’s clefts. We identified two key clusters that may function in the loading and unloading of PfEMP1 into and out of the Maurer’s clefts. We focus on a putative PfEMP1 loading complex that includes the protein GEXP07/CX3CL1-binding protein 2 (CBP2). Disruption of GEXP07 causes Maurer’s cleft fragmentation, aberrant knobs, ablation of PfEMP1 surface expression, and loss of the PfEMP1-mediated adhesion. ΔGEXP07 parasites have a growth advantage compared to wild-type parasites, and the infected RBCs are more deformable and more osmotically fragile. IMPORTANCE The trafficking of the virulence antigen PfEMP1 and its presentation at the knob structures at the surface of parasite-infected RBCs are central to severe adhesion-related pathologies such as cerebral and placental malaria. This work adds to our understanding of how PfEMP1 is trafficked to the RBC membrane by defining the protein-protein interaction networks that function at the Maurer’s clefts controlling PfEMP1 loading and unloading. We characterize a protein needed for virulence protein trafficking and provide new insights into the mechanisms for host cell remodeling, parasite survival within the host, and virulence.

Ovine footrot is a degenerative disease of sheep feet leading to the separation of hoof-horn from the underlying skin and lameness. This study quantitatively examined histological features of the ovine interdigital skin as well as their relationship with pro-inflammatory cytokine (IL-1β) and virulent Dichelobacter nodosus in footrot. From 55 healthy and 30 footrot ovine feet, parallel biopsies (one fixed for histology) were collected post-slaughter and analysed for lesions and histopathological analysis using haematoxylin and eosin and Periodic Acid-Schiff. Histological lesions were similar in both conditions while inflammatory scores mirror IL-1β expression levels. Increased inflammatory score corresponded with high virulent D. nodosus load and was significant (p

The Pragmatic Insertional Mutation Mapping (PIMMS) laboratory protocol was developed alongside various bioinformatics packages (Blanchard et al 2015) to enable detection of essential and conditionally essential genes in Streptococcus and related bacteria. This extended the methodology commonly used to locate insertional mutations in individual mutants to the analysis of mutations in populations of bacteria. In S. uberis, a pyogenic Streptococcus associated with intramammary infection and mastitis in ruminants, the mutagen pGhost9:ISS1 was shown to integrate across the entire genome. Analysis of >80,000 mutations revealed 196 coding sequences, which were not be mutated and a further 67 where mutation only occurred beyond the 90th percentile of the coding sequence. These sequences showed good concordance with sequences within the database of essential genes (DEG) and typically matched sequences known to be associated with basic cellular functions. Due to the broad utility of this mutagen and the simplicity of the methodology it is anticipated that PIMMS will be of value to a wide range of laboratories in functional genomic analysis of a wide range of Gram positive bacteria (Streptococcus, Enterococcus and Lactococcus) of medical, veterinary and industrial significance.