A novel acidification mechanism for greatly enhanced oxygen supply to the fish retina.

Previously, we showed that the evolution of high acuity vision in fishes was directly associated with their unique pH-sensitive hemoglobins that allow O2 to be delivered to the retina at PO2s more than ten-fold that of arterial blood (Damsgaard et al., 2019). Here, we show strong evidence that vacuolar-type H+-ATPase and plasma-accessible carbonic anhydrase in the vascular structure supplying the retina act together to acidify the red blood cell leading to O2 secretion. In vivo data indicate that this pathway primarily affects the oxygenation of the inner retina involved in signal processing and transduction, and that the evolution of this pathway was tightly associated with the morphological expansion of the inner retina. We conclude that this mechanism for retinal oxygenation played a vital role in the adaptive evolution of vision in teleost fishes.

Retinal oxygen supply shaped the functional evolution of the vertebrate eye.

The retina has a very high energy demand but lacks an internal blood supply in most vertebrates. Here we explore the hypothesis that oxygen diffusion limited the evolution of retinal morphology by reconstructing the evolution of retinal thickness and the various mechanisms for retinal oxygen supply, including capillarization and acid-induced haemoglobin oxygen unloading. We show that a common ancestor of bony fishes likely had a thin retina without additional retinal oxygen supply mechanisms and that three different types of retinal capillaries were gained and lost independently multiple times during the radiation of vertebrates, and that these were invariably associated with parallel changes in retinal thickness. Since retinal thickness confers multiple advantages to vision, we propose that insufficient retinal oxygen supply constrained the functional evolution of the eye in early vertebrates, and that recurrent origins of additional retinal oxygen supply mechanisms facilitated the phenotypic evolution of improved functional eye morphology.

Postmortem protein stability investigations of the human hepatic drug-metabolizing cytochrome P450 enzymes CYP1A2 and CYP3A4 using mass spectrometry.

Variability in expression and activity of hepatic drug-metabolizing cytochrome P450 (CYP) enzymes can play a causal role in fatal intoxication cases and is thus of forensic interest. We investigated the feasibility of LC-MS/MS based quantification and in vitro enzyme activity measurements of two major drug-metabolizing enzymes CYP1A2 and CYP3A4 in postmortem human liver microsomes (HLM). In autopsy cases (postmortem interval 24-36 h) we found CYP1A2 and CYP3A4 protein levels similar to that measured in a non-decayed reference HLM pool, whereas CYP1A2 and CYP3A4 enzyme activities were absent or severely decreased. Stability studies showed that CYP1A2 and CYP3A4 protein abundances were relatively stable in tissue stored in vitro for up to seven days at 4 °C. When tissue was stored for more than one day at 21 °C variable and case-specific decay patterns were observed, and CYP abundances declined especially after 3-4 days storage. Investigations of 50 autopsy cases revealed mean CYP1A2 and CYP3A4 levels of 49 and 47 pmol per mg HLM protein and inter-individual variabilities similar to those reported in other studies. This study supports postmortem quantification of CYP proteins in autopsy hepatic tissue by mass spectrometry. SIGNIFICANCE: This study indicates that MS-based detection of drug-metabolizing cytochrome P450 (CYP) proteins is achievable in postmortem hepatic tissue and that acceptable quantification data are obtainable but dependent on the storage conditions and postmortem sampling time. CYP abundance data could contribute to a conceivable way of assessing individual CYP activity phenotypes in a postmortem context.

DNA and RNA analysis of blood and muscle from bodies with variable postmortem intervals.

The breakdown of DNA and RNA in decomposing human tissue represents a major obstacle for postmortem forensic molecular analysis. This study investigated the feasibility of performing PCR-based molecular analysis of blood and muscle tissue from 45 autopsy cases with defined postmortem intervals ranging from one to more than 14 days. It was not possible to collect blood from 38 % of the autopsy cases due to severe coagulation and hemolysis, whereas muscle tissue was available for all cases. PCR-amplifiable DNA could be extracted from 96 % of the frozen muscle specimens and from 93 % of the formalin fixed and paraffin embedded (FFPE) muscle specimens. A quality assessment of muscle-derived DNA showed increased fragmentation with advancing body decomposition and generally more fragmentation in DNA from FFPE tissue than in DNA from frozen tissue. It was possible to amplify 1,000 basepair (bp) DNA fragments from all samples with postmortem intervals below 3 days whereas 400-600 bp long fragments typically could be amplified from the most decomposed muscle specimens. RNA was less stable than DNA in postmortem muscle tissue, yet selected mRNA molecules could be detected by reverse-transcriptase PCR in all samples up to 3 days after death. We conclude that analysis of DNA from bodies with a wide postmortem interval range is usually possible whereas the consistency of RNA analyses decreases considerably 3 days postmortem. We showed that muscle tissue is a highly usable source of DNA and RNA for postmortem forensic molecular analysis as well as for retrospective research projects based on archived FFPE specimens.

Expression level, tissue distribution pattern, and prognostic impact of vascular endothelial growth factors VEGF and VEGF-C and their receptors Flt-1, KDR, and Flt-4 in different subtypes of non-Hodgkin lymphomas.

The aim of the study was to investigate the expression of angio- and lymphangiogenic molecules (vascular endothelial growth factors VEGF and VEGF-C and their receptors Flt-1, KDR, and Flt-4) in non-Hodgkin lymphomas (NHL) treated in the pre-rituximab era. Pre-therapeutic lymph-node biopsies from 155 patients with NHL (64 follicular lymphomas (FLs), 47 de novo diffuse large B-cell lymphomas (DLBCL) and 44 peripheral T-cell lymphomas (PTCL)) were stained by in situ hybridization and immunohistochemistry. Tumor cell expression of VEGF, VEGF-C and their receptors was detected in most of the analyzed biopsies. In FL, diffuse intratumoral VEGF staining correlated with shorter overall survival (OS) (p = 0.008) and diffuse KDR staining was associated with a higher risk of histologic transformation (p = 0.05). In DLBCL, high KDR expression predicted poor treatment response (p = 0.03) and had a significant adverse impact on OS (p < 0.001). In PTCL, diffuse tissue distribution of VEGF mRNA correlated with an unfavorable 5-year OS (p = 0.004).

Immunohistochemical Estimates of Angiogenesis, Proliferative Activity, p53 Expression, and Multiple Drug Resistance Have No Prognostic Impact in Osteosarcoma: A Comparative Clinicopathological Investigation.

Purpose. To investigate angiogenesis, multiple drug resistance (MDR) and proliferative activity as prognostic variables in patients suffering from osteosarcoma. Methods. Histologic biopsies from 117 patients treated in the period from 1972 through 1999 were immunohistologically investigated regarding angiogenesis (CD34), proliferative activity (MIB-1), and the expression of p53 and MDR (P-glycoprotein (Pgp); clones JSB-1, C494, and MRK16). Quantitative and semiquantitative scores of immunoreactive cells were analyzed statistically along with retrospectively obtained clinicopathologic variables. Results. Chemotherapy reduced the rate of amputations (P = .00002). The Pgp was overexpressed (score >/=2) in 48% of the primary, diagnostic biopsies, and high Pgp correlated with high Pgp in postsurgical specimens (P = .003). In contrast, no such associations were disclosed for estimates of angiogenesis (P = .64) and p53 (P > .32), whereas the MIB-1 index was reduced in the post-chemotherapy specimens (P = .02). The overall, disease-specific survival was 47%, increasing to 54% in patients receiving pre-operative chemotherapy. Statistical analyses showed prognostic impact exclusively by patient age and type of osteosarcoma. Discussion. The studied series of patients documented already prior to the chemotherapy era, a rather excellent survival and estimates of angiogenesis, proliferation, p53, and Pgp expressions, did not demonstrate sufficient power to serve as predictors of treatment response or survival.