Myosin II synergizes with F-actin to promote DNGR-1-dependent cross-presentation of dead cell-associated antigens

Conventional type 1 DCs (cDC1s) excel at cross-presentation of dead cell-associated antigens partly because they express DNGR-1, a receptor that recognizes exposed actin filaments on dead cells. In vitro polymerized F-actin can be used as a synthetic ligand for DNGR-1. However, cellular F-actin is decorated with actin-binding proteins, which could affect DNGR-1 recognition. Here, we demonstrate that myosin II, an F-actin-associated motor protein, greatly potentiates the binding of DNGR-1 to F-actin. Latex beads coated with F-actin and myosin II are taken up by DNGR-1+ cDC1s, and antigen associated with those beads is efficiently cross-presented to CD8+ T cells. Myosin II-deficient necrotic cells are impaired in their ability to stimulate DNGR-1 or to serve as substrates for cDC1 cross-presentation to CD8+ T cells. These results provide insights into the nature of the DNGR-1 ligand and have implications for understanding immune responses to cell-associated antigens and for vaccine design

P–042 Impact of semen parameters, sperm DNA fragmentation and sperm aneuploidy in male infertility

Study question. Should sperm aneuploidies and sperm DNA fragmentation (sDNAfrag) be included as valid tests in the routine investigation of male infertility? Summary answer. Sperm DNA fragmentation was associated with male age, oligozoospermia (OZ), oligoteratozoospermia (OT), astenoteratozoospermia (AT) and oligoastenoteratozoospermia (OAT). Sperm aneuploidies were associated with OT and OAT. What is known already. Semen parameters assist male infertility diagnosis and treatment, but sDNAfrag and aneuploidy analysis could add useful information, as abnormal values compromise fertility. To include these tests in the routine diagnosis it should be determined if behave as informative parameter and add information regarding the fertility status. For that, further studies comparing these tests to semen parameters are needed, since previous results are not consensual. Additionally, standardization of a sDNAfrag cut-off is needed, as different sample sizes and techniques originate distinct results. Also, until a standardization of the protocol is missing, a cut-off value should be defined for each laboratory. Study design, size, duration. A retrospective and prospective investigation was performed, within a 12 years period (April 2007-December 2019). A total of 835 infertile males with a normal karyotype (46,XY) were included. Karyotyping and evaluation of sDNAfrag and sperm aneuploidies were made at a public Genetic unit. All normozoospermic (NZ) patients with a born child and patients whose infertility treatments were done due to female factors were selected from our database and used as controls (60 individuals). Participants/materials, setting, methods. Semen analysis followed WHO–2010 guidelines. sDNAfrag was evaluated using the TUNEL assay. Sperm aneuploidies were detected using FISH (chromosomes 13, 18, 21, X, Y). Several tests were applied: correlations for linear associations between numerical variables, ANOVA for comparisons between means, Dunn-test for post-hoc comparisons. To determine the sDNAfrag cut-off value, the area under the ROC curve, sensitivity and specificity, were calculated, with the Youden-Index used to find a threshold that maximizes both sensitivity and specificity. Main results and the role of chance. Regarding male age, it was observed a positive correlation with sperm concentration, a negative correlation with sperm vitality (VT) and hypoosmolality, and a positive correlation with sDNAfrag. Regarding sDNAfrag, it was observed negative correlation with sperm concentration, total progressive motility (TPM), morphology, VT and hypoosmolality. Regarding sperm aneuploidies, both total sperm aneuploidy and total sperm disomy exhibited a negative association with sperm concentration, TPM and morphology. It was also investigated whose groups of individuals could be indicated for sDNAfrag or sperm aneuploidy testing. The NZ group evidenced significant lower sDNAfrag, total sperm aneuploidy and total sperm disomy in relation to the non-NZ group. In the NZ group, sDNAfrag was significantly lower in relation to the OZ, OT, AT and OAT groups. The NZ group presented significant lower percentages of sperm aneuploidy in relation to the OT and OAT groups, and significant lower percentages of sperm disomy in relation to the OAT group. Additionally, sDNAfrag was positively correlated with total sperm aneuploidy and total sperm disomy. From the present large population, ROC curve analysis allowed estimating a cut-off value of 18.8% for the TUNEL-assay (sDNAfrag), with 0.658 of area under the curve, 53.9% sensitivity and 76.7% specificity. Limitations, reasons for caution. Although presenting a high number of cases and strict controls, the present study was unable to include as controls healthy men with proven fertility. Additionally, the present study did not take into account life-style factors and male associated pathologies besides infertility. Wider implications of the findings: Semen parameters were shown to be negatively correlated with sDNAfrag and sperm aneuploidies. As sDNAfrag testing and sperm aneuploidy testing were associated with semen abnormalities and male age, it is suggested their inclusion in the routine evaluation of infertile men, thus adding important complementary information about the fertility status

Flow of red blood cells suspensions through hyperbolic microcontractions

The present study uses a hyperbolic microchannel with a low aspect ratio (AR) to investigate how the red blood cells (RBCs) deform under conditions of both extensional and shear induced flows. The deformability is presented by the degree of the deformation index (DI) of the flowing RBCs throughout the microchannel at its centerline. A suitable image analysis technique is used for semi-automatic measurements of average DIs, velocity and strain rate of the RBCs travelling in the regions of interest. The results reveal a strong deformation of RBCs under both extensional and shear stress dominated flow conditions

The application of Bonelike® Poro as a synthetic bone substitute for the management of critical-sized bone defects - A comparative approach to the autograft technique - A preliminary study

The effective treatment of non-unions and critical-sized defects remains a challenge in the orthopedic field. From a tissue engineering perspective, this issue can be addressed through the application bioactive matrixes to support bone regeneration, such as Bonelike®, as opposed to the widespread autologous grafting technique. An improved formulation of Bonelike® Poro, was assessed as a synthetic bone substitute in an ovine model for critical-sized bone defects. Bone regeneration was assessed after 5 months of recovery through macro and microscopic analysis of the healing features of the defect sites. Both the application of natural bone graft or Bonelike® Poro resulted in bridging of the defects margins. Untreated defect remained as fibrous non-unions at the end of the study period. The characteristics of the newly formed bone and its integration with the host tissue were assessed through histomorphometric and histological analysis, which demonstrated Bonelike® Poro to result in improved healing of the defects. The group treated with synthetic biomaterial presented bone bridges of increased thickness and bone features that more closely resembled the native spongeous and cortical bone. The application of Bonelike® Poro enabled the regeneration of critical-sized lesions and performed comparably to the autograph technique, validating its octeoconductive and osteointegrative potential for clinical application as a therapeutic strategy in human and veterinary orthopedics.This research was supported by Projects PEst-OE/AGR/UI0211/2011 from FCT , and COMPETE 2020 , from ANI – Projetos ID&T Empresas em Copromoção , by the project “insitu.Biomas – Reinvent biomanufacturing systems by using an usability approach for in situ clinic temporary implants fabrication” with the reference POCI-01-0247-FEDER-017771 , by the project “Print-on-Organs – Engineering bioinks and processes for direct printing on organs” with the reference POCI-01-0247-FEDER-033877 , and by the project “Bone2Move - Development of ‘in vivo’ experimental techniques and modelling methodologies for the evaluation of 4D scaffolds for bone defect in sheep model: an integrative research approach” with the reference POCI-01-0145-FEDER-031146 . Mariana Vieira Branquinho ( SFRH/BD/146172/2019 ), Ana Catarina Sousa ( SFRH/BD/146689/2019 ), and Rui Damásio Alvites ( SFRH/BD/116118/2016 ), acknowledge FCT , for financial support. This research was supported by Projects PEst-OE/AGR/UI0211/2011 from FCT, and COMPETE 2020, from ANI ? Projetos ID&T Empresas em Copromo??o, by the project ?insitu.Biomas ? Reinvent biomanufacturing systems by using an usability approach for in situ clinic temporary implants fabrication? with the reference POCI-01-0247-FEDER-017771, by the project ?Print-on-Organs ? Engineering bioinks and processes for direct printing on organs? with the reference POCI-01-0247-FEDER-033877, and by the project ?Bone2Move - Development of ?in vivo? experimental techniques and modelling methodologies for the evaluation of 4D scaffolds for bone defect in sheep model: an integrative research approach? with the reference POCI-01-0145-FEDER-031146. Mariana Vieira Branquinho (SFRH/BD/146172/2019), Ana Catarina Sousa (SFRH/BD/146689/2019), and Rui Dam?sio Alvites (SFRH/BD/116118/2016), acknowledge FCT, for financial support