Role of myeloid cells in ischemic retinopathies: recent advances and unanswered questions.

Myeloid cells including microglia and macrophages play crucial roles in retinal homeostasis by clearing cellular debris and regulating inflammation. These cells are activated in several blinding ischemic retinal diseases including diabetic retinopathy, where they may exert both beneficial and detrimental effects on neurovascular function and angiogenesis. Myeloid cells impact the progression of retinal pathologies and recent studies suggest that targeting myeloid cells is a promising therapeutic strategy to mitigate diabetic retinopathy and other ischemic retinal diseases. This review summarizes the recent advances in our understanding of the role of microglia and macrophages in retinal diseases and focuses on the effects of myeloid cells on neurovascular injury and angiogenesis in ischemic retinopathies. We highlight gaps in knowledge and advocate for a more detailed understanding of the role of myeloid cells in retinal ischemic injury to fully unlock the potential of targeting myeloid cells as a therapeutic strategy for retinal ischemia.

Evolution of immunogenetic components encoding ultralong CDR H3.

The genomes of most vertebrates contain many V, D, and J gene segments within their Ig loci to construct highly variable CDR3 sequences through combinatorial diversity. This nucleotide variability translates into an antibody population containing extensive paratope diversity. Cattle have relatively few functional VDJ gene segments, requiring innovative approaches for generating diversity like the use of ultralong-encoding IGHV and IGHD gene segments that yield dramatically elongated CDR H3. Unique knob and stalk microdomains create protracted paratopes, where the antigen-binding knob sits atop a long stalk, allowing the antibody to bind both surface and recessed antigen epitopes. We examined genomes of twelve species of Bovidae to determine when ultralong-encoding IGHV and IGHD gene segments evolved. We located the 8-bp duplication encoding the unique TTVHQ motif in ultralong IGHV segments in six Bovid species (cattle, zebu, wild yak, domestic yak, American bison, and domestic gayal), but we did not find evidence of the duplication in species beyond the Bos and Bison genera. Additionally, we analyzed mRNA from bison spleen and identified a rich repertoire of expressed ultralong CDR H3 antibody mRNA, suggesting that bison use ultralong IGHV transcripts in their host defense. We found ultralong-encoding IGHD gene segments in all the same species except domestic yak, but again not beyond the Bos and Bison clade. Thus, the duplication event leading to this ultralong-encoding IGHV gene segment and the emergence of the ultralong-encoding IGHD gene segment appears to have evolved in a common ancestor of the Bos and Bison genera 5-10 million years ago.

Comparative study of cartilaginous fish divulges insights into the early evolution of primary, secondary and mucosal lymphoid tissue architecture.

Cartilaginous fish are located at a pivotal point in phylogeny where the adaptive immune system begins to resemble that of other, more-derived jawed vertebrates, including mammals. For this reason, sharks and other cartilaginous fish are ideal models for studying the natural history of immunity. Insights from such studies may include distinguishing the (evolutionarily conserved) fundamental aspects of adaptive immunity from the (more recent) accessory. Some lymphoid tissues of sharks, including the thymus and spleen, resemble those of mammals in both appearance and function. The cartilaginous skeleton of sharks has no bone marrow, which is also absent in bony fish despite calcified bone, but cartilaginous fish have other Leydig's and epigonal organs that function to provide hematopoiesis analogous to mammalian bone marrow. Conserved across all vertebrate phylogeny in some form is gut-associated lymphoid tissues, or GALT, which is seen from agnathans to mammals. Though it takes many forms, from typhlosole in lamprey to Peyer's patches in mammals, the GALT serves as a site of antigen concentration and exposure to lymphocytes in the digestive tract. Though more complex lymphoid organs are not present in agnathans, they have several primitive tissues, such as the thymoid and supraneural body, that appear to serve their variable lymphocyte receptor-based adaptive immune system. There are several similarities between the adaptive immune structures in cartilaginous and bony fish, such as the thymus and spleen, but there are mechanisms employed in bony fish that in some instances bridge their adaptive immune systems to that of tetrapods. This review summarizes what we know of lymphoid tissues in cartilaginous fishes and uses these data to compare primary and secondary tissues in jawless, cartilaginous, and bony fishes to contextualize the early natural history of vertebrate mucosal immune tissues.

Implementing a Hybrid Expression Method That Allows Upper-Division Biochemistry Lab Students To Engage in a Full Protein Production Experience While Allowing Ample Time for Characterization Experiments.

Protein structure, function, and signaling are a large portion of biochemistry. Because of this, proteins are often used as model systems in biochemistry laboratory courses, where a course-long project might comprise protein expression, purification, and characterization. Two common protein expression methods are isopropyl ß-d-1-thiogalactopyranoside (IPTG) induction, which utilizes easy-to-make media but requires extensive cell-growth monitoring that is time-intensive, and autoinduction, which employs multicomponent media that are time-consuming to make but require no cell-growth monitoring. A protein expression method that is a hybrid of IPTG induction and autoinduction is presented. The hybrid method utilizes the medium of IPTG induction and the no-cell-growth-monitoring induction process of autoinduction, saving hands-on time in the protein expression phase to allow more time for protein characterization while still having students execute each step.