Iron and Porphyrin Trafficking in Heme Biogenesis

Iron is an essential element for diverse biological functions. In mammals, the majority of iron is enclosed within a single prosthetic group: heme. In metazoans, heme is synthesized via a highly conserved and coordinated pathway within the mitochondria. However, iron is acquired from the environment and subsequently assimilated into various cellular pathways, including heme synthesis. Both iron and heme are toxic but essential cofactors. How is iron transported from the extracellular milieu to the mitochondria? How are heme and heme intermediates coordinated with iron transport? Although recent studies have answered some questions, several pieces of this intriguing puzzle remain unsolved.     

Colonization factors of diarrheagenicE. coli and their intestinal receptors

WhileEscherichia coli is common as a commensal organism in the distal ileum and colon, the presence of colonization factors (CF) on pathogenic strains ofE. coli facilitates attachment of the organism to intestinal receptor molecules in a species- and tissue-specific fashion. After the initial adherence, colonization occurs, and the involvement of additional virulence determinants leads to illness. EnterotoxigenicE. coli (ETEC) is the most extensively studied of the five categories ofE. coli that cause diarrheal disease, and has the greatest impact on health worldwide. ETEC can be isolated from domestic animals and humans. The biochemistry, genetics, epidemiology, antigenic characteristics, and cell and receptor binding properties of ETEC have been extensively described. Another major category, enteropathogenicE. coli (EPEC), has virulence mechanisms, primarily effacement and cytoskeletal rearrangement of intestinal brush borders, that are distinct from ETEC. An EPEC CF receptor has been purified and characterized as a sialidated transmembrane glycoprotein complex directly attached to actin, thereby associating CF-binding with host-cell response. Three, additional categories ofE. coli diarrheal disease, their colonization factors and their host cell receptors are discussed. It appears that biofilms exist in the intestine in a manner similar to oral bacterial biofilms, and thatE. coli is part of these biofilms as both commensals and pathogens.Abbreviations CF colonization factor - CFA Colonization Factor Antigen - CS coli-surface-associated antigen - EAggEC enteroaggregativeE. coli - ECDD E. coli diarrheal disease - EHEC enterohemorrhagicE. coli - EIEC enteroinvasiveE. coli - EPEC enteropathogenicE. coli - ETEC enterotoxigenicE. coli - Gal galactose - GalNAc N-acetyl galactosamine - LT heat-labile toxin - NeuAc N-acetyl neuraminic acid - PCF Putative colonization factor - RBC red blood cells - SLT Shiga-like toxin - ST heat-stable toxin     

The Codon Usage in the Minimal Natural Cell

A statistical analysis of the variation in contents with the size of the current known smallest genomes, N. deltocephalinicola, C. ruddii, N. equitans, and M. genitalium, enabled the indication of a minimal set of codons capable of naturally building a modern-type free-living unicellular organism in an early stage of evolution. Using a linear regression model, the potential codon distribution in the minimal natural cell was predicted and compared to the composition of the smallest synthetic, JCVI-Syn3.0. The distribution of the molecular weight of potentially coded amino acids was also calculated. The main differences in the features of the minimal natural cell and H. Sapiens genome were analyzed. In this regard, the content percentage of respective amino acids and their polarization charge properties were reported and compared. The fractions of occurring nucleotides were calculated, too. Then, the estimated numbers of codons in a minimal natural cell were related to the expected numbers for random distribution. Shown increase, or decrease in the contents, relative to the calculated random filling was related to the evolutionary preferences, varying with the subsequent eras of the evolution of genetic code.

     

Split-wrmScarlet and split-sfGFP: tools for faster,easier fluorescent labeling of endogenous proteins in Caenorhabditis elegans

We create and share a new red fluorophore, along with a set of strains, reagents and protocols, to make it faster and easier to label endogenous Caenorhabditis elegans proteins with fluorescent tags. CRISPR-mediated fluorescent labeling of C. elegans proteins is an invaluable tool, but it is much more difficult to insert fluorophore-size DNA segments than it is to make small gene edits. In principle, high-affinity asymmetrically split fluorescent proteins solve this problem in C. elegans: the small fragment can quickly and easily be fused to almost any protein of interest, and can be detected wherever the large fragment is expressed and complemented. However, there is currently only one available strain stably expressing the large fragment of a split fluorescent protein, restricting this solution to a single tissue (the germline) in the highly autofluorescent green channel. No available C. elegans lines express unbound large fragments of split red fluorescent proteins, and even state-of-the-art split red fluorescent proteins are dim compared to the canonical split-sfGFP protein. In this study, we engineer a bright, high-affinity new split red fluorophore, split-wrmScarlet. We generate transgenic C. elegans lines to allow easy single-color labeling in muscle or germline cells and dual-color labeling in somatic cells. We also describe a novel expression strategy for the germline, where traditional expression strategies struggle. We validate these strains by targeting split-wrmScarlet to several genes whose products label distinct organelles, and we provide a protocol for easy, cloning-free CRISPR/Cas9 editing. As the collection of split-FP strains for labeling in different tissues or organelles expands, we will post updates at doi.org/10.5281/zenodo.3993663     

Determination of acid α-naphthyl acetate esterase enzyme activity in peripheral blood leukocytes of gazelles (Gazella subgutturosa)

We examined gazelle peripheral blood leucocytes using the α-Naphthyl acetate esterase (ANAE) staining technique (pH 5.8). Our purpose was to determine the percentage of ANAE positive lymphocytes. The proportion of ANAE positive T-lymphocytes was 72%. T-lymphocytes showed an ANAE positive reaction, but eosinophilic granulocytes and monocytes also showed a positive reaction. By contrast, no reaction was detected in B-lymphocytes, neutrophil granulocytes or platelets. The reaction observed in T-lymphocytes was a red-brown coloration, usually 1–2 granules, but enough granules to fill the cytoplasm were detected rarely. As a result of ANAE enzyme staining, we concluded that the staining technique can be used as a cytochemical marker for gazelle T-lymphocytes.     

The use of compensatory base change analysis of ITS2 as a tool in the phylogeny of Mucorales, illustrated by the Mucor circinelloides complex

Compensatory base changes (CBCs) in helix II of rDNA ITS2, suggested as a molecular classifier for fungi, were analyzed in Mucor circinelloides and its varieties. Only a few CBCs were found in the complex. Three out of the four accepted formae (f. circinelloides, f. lusitanicus, f. janssenii) did not exhibit CBCs. One CBC was found between strains that form zygospores; consequently, CBC is not always concordant with mating experiments. Strains with two CBC are unable to breed. It is suggested that some strains of the M. circinelloides complex are at the beginning of speciation.     

The Comparison of the Effects of Three Physiotherapy Techniques on Hamstring Flexibility in Children: A Prospective,Randomized, Single-Blind Study

The aim of the study was to evaluate changes in hamstring flexibility in 120 asymptomatic children who participated in a 6-week program consisting of one physiotherapy session per week and daily home exercises. The recruitment criteria included age (10–13 years), no pain, injury or musculoskeletal disorder throughout the previous year, physical activity limited to school sport. Subjects were randomly assigned to one of the three groups: (1) post-isometric relaxation – PIR (n = 40), (2) static stretch combined with stabilizing exercises – SS (n = 40) and (3) stabilizing exercises – SE (n = 40). Hamstring flexibility was assessed with straight leg raise (SLR), popliteal angle (PA) and finger-to-floor (FTF) tests. The examinations were conducted by blinded observers twice, prior to the program and a week after the last session with the physiotherapist. Twenty-six children who did not participate in all six exercise sessions with physiotherapists were excluded from the analysis. The results obtained by 94 children were analyzed (PIR, n = 32; SS, n = 31; SE, n = 31). In the PIR and SS groups, a significant (P<0.01) increase in SLR, PA, FTF results was observed. In the SE group, a significant (P<0.001) increase was observed in the SLR but not in the PA and FTF (P>0.05). SLR result in the PIR and SS groups was significantly (P<0.001) higher than in the SE group. As far as PA results are concerned, a significant difference was observed only between the SS and SE groups (P = 0.014). There were no significant (P = 0.15) differences regarding FTF results between the three groups. Post-isometric muscle relaxation and static stretch with stabilizing exercises led to a similar increase in hamstring flexibility and trunk forward bend in healthy 10–13-year-old children. The exercises limited to straightening gluteus maximus improved the SLR result, but did not change the PA and FTF results.