Treponema pallidum 3-phosphoglycerate mutase is a heat-labile enzyme that may limit the maximum growth temperature for the spirochete

In the causative agent of syphilis, Treponema pallidum, the gene encoding 3-phosphoglycerate mutase, gpm, is part of a six-gene operon (tro operon) that is regulated by the Mn-dependent repressor TroR. Since substrate-level phosphorylation via the Embden-Meyerhof pathway is the principal way to generate ATP in T. pallidum and Gpm is a key enzyme in this pathway, Mn could exert a regulatory effect on central metabolism in this bacterium. To study this, T. pallidum gpm was cloned, Gpm was purified from Escherichia coli, and antiserum against the recombinant protein was raised. Immunoblots indicated that Gpm was expressed in freshly extracted infective T. pallidum. Enzyme assays indicated that Gpm did not require Mn(2+) while 2,3-diphosphoglycerate (DPG) was required for maximum activity. Consistent with these observations, Mn did not copurify with Gpm. The purified Gpm was stable for more than 4 h at 25 degrees C, retained only 50% activity after incubation for 20 min at 34 degrees C or 10 min at 37 degrees C, and was completely inactive after 10 min at 42 degrees C. The temperature effect was attenuated when 1 mM DPG was added to the assay mixture. The recombinant Gpm from pSLB2 complemented E. coli strain PL225 (gpm) and restored growth on minimal glucose medium in a temperature-dependent manner. Increasing the temperature of cultures of E. coli PL225 harboring pSLB2 from 34 to 42 degrees C resulted in a 7- to 11-h period in which no growth occurred (compared to wild-type E. coli). These data suggest that biochemical properties of Gpm could be one contributing factor to the heat sensitivity of T. pallidum.     

Interactions ofCandida albicans with bacteria and salivary molecules in oral biofilms

The yeastCandida albicans coaggregates with a variety of streptococcal species, an interaction that may promote oral colonization by yeast cells.C. albicans andCandida tropicalis are the yeasts most frequently isolated from the human oral cavity and our data demonstrate that both these species bind toStreptococcus gordonii NCTC 7869 while two otherCandida species (Candida krusei andCandida kefyr) do not. Adherence ofC. albicans was greatest when the yeast had been grown at 30° C to mid-exponential growth phase. For 21 strains ofC. albicans there was a positive correlation between the ability to adhere toS. gordonii and adherence to experimental salivary pellicle. Whole saliva either stimulated or slightly inhibited adherence ofC. albicans toS. gordonii depending on the streptococcal growth conditions. The results suggest that the major salivary adhesins and coaggregation adhesins ofC. albicans are co-expressed.     

DNA recognition properties of the N-terminal DNA binding domain within the large subunit of replication factor C.

Replication Factor C (RFC) is a five-subunit protein complex required for eukaryotic DNA replication and repair. The large subunit within this complex contains a C-terminal DNA binding domain which provides specificity for PCNA loading at a primer-template and a second, N-terminal DNA binding domain of unknown function. We isolated the N-terminal DNA binding domain from Drosophila melanogaster and defined the region within this polypeptide required for DNA binding. The DNA determinants most efficiently recognized by both the Drosophila minimal DNA binding domain and the N-terminal half of the human large subunit consist of a double-stranded DNA containing a recessed 5' phosphate. DNA containing a recessed 5' phosphate was preferred 5-fold over hairpined DNA containing a recessed 3' hydroxyl. Combined with existing data, these DNA binding properties suggest a role for the N-terminal DNA binding domain in the recognition of phosphorylated DNA ends.     

Insertion of a reversible redox switch into a rare-cutting DNA endonuclease

Target sites for homing endonucleases occur infrequently in complex genomes. As a consequence, these enzymes can be used in mammalian systems to introduce double-strand breaks at recognition sites inserted within defined loci to study DNA repair by homologous and nonhomologous recombination. Using homing endonucleases for gene targeting in vivo would be more feasible if temporal or spatial regulation of their enzymatic activity were possible. Here, we show that the DNA cleavage activity of the yeast PI-SceI homing endonuclease can be turned on and off using a redox switch. Two cysteine pairs (Cys-64/Cys-344 and Cys-67/Cys-365) were separately inserted into flexible DNA binding loop(s) to create disulfide bonds that lock the endonuclease into a nonproductive conformation. The cleavage activities of the reduced Cys-64/Cys-344 and Cys-67/Cys-365 variants are similar or slightly lower than that of the control protein, but the activities of the proteins in the oxidized state are decreased more than 30-fold. Modulating the activity of the proteins is easily accomplished by adding or removing the reducing agent. We show that defects in DNA binding account for the decreased DNA cleavage activities of the proteins containing disulfide bonds. Interestingly, the Cys-67/Cys-365 variant toggles between two different DNA binding conformations under reducing and oxidizing conditions, which may permit the identification of structural differences between the two states. These studies demonstrate that homing endonuclease activity can be controlled using a molecular switch.     

Repercussions of the COVID-19 pandemic on athletes: a cross-sectional study

The COVID-19 pandemic has presented significant challenges and implications for the sports community. Thus, this study aimed to describe the prevalence of COVID-19 in Brazilian athletes and identify the epidemiological, clinical, athletic, life and health factors associated with the disease in these individuals. A cross-sectional study was performed involving 414 athletes from 22 different sports using an online questionnaire from August to November 2020. The association between the athletes’ characteristics and COVID-19 was evaluated using a logistic regression model. The prevalence of COVID-19 was 8.5%, although only 40% of athletes reported having been tested. Being under 27 years of age (3-fold), having children (~5-fold), having a teammate test positive for COVID-19 (2.5-fold), and smoking (14-fold) were associated with a possible higher risk of disease. Almost 20% of athletes self-reported musculoskeletal injuries during the period of the pandemic that was studied. Athletes with a university education (P = 0.02), a profession other than sports (P < 0.001), those from a low-income family (P = 0.01), and public health system users (P = 0.04) were significantly less frequently tested for COVID-19, whereas international competitors, athletes who received a wage, and athletes who had a teammate who tested positive for COVID-19 were 2-, 3-, and 15-fold more likely to be tested for COVID-19, respectively. Approximately 26% of the athletes who tested negative or were untested reported more than three characteristic COVID-19 symptoms, and 11% of athletes who tested positive for COVID-19 were asymptomatic. The identification of modifiable (have children, smoking, and teammates positively tested) and non-modifiable (age under 27 years) factors related to COVID-19 in athletes can contribute to implementing surveillance programmes to decrease the incidence of COVID-19 in athletes and its negative impacts in sports.     

Deletion of sterol O-acyltransferase 2 (SOAT2) function in mice deficient in lysosomal acid lipase (LAL) dramatically reduces esterified cholesterol sequestration in the small intestine and liver

Sterol O-acyltransferase 2 (SOAT2), also known as ACAT2, is the major cholesterol esterifying enzyme in the liver and small intestine (SI). Esterified cholesterol (EC) carried in certain classes of plasma lipoproteins is hydrolyzed by lysosomal acid lipase (LAL) when they are cleared from the circulation. Loss-of-function mutations in LIPA, the gene that encodes LAL, result in Wolman disease (WD) or cholesteryl ester storage disease (CESD). Hepatomegaly and a massive increase in tissue EC levels are hallmark features of both disorders. While these conditions can be corrected with enzyme replacement therapy, the question arose as to what effect the loss of SOAT2 function might have on tissue EC sequestration in LAL-deficient mice. When weaned at 21 days, Lal^−/−:Soat2^+/+ mice had a whole liver cholesterol content (mg/organ) of 24.7 mg vs 1.9 mg in Lal^+/+:Soat2^+/+ littermates, with almost all the excess sterol being esterified. Over the next 31 days, liver cholesterol content in the Lal^−/−:Soat2^+/+ mice increased to 145 ± 2 mg but to only 29 ± 2 mg in their Lal^−/−:Soat2^−/− littermates. The level of EC accumulation in the SI of the Lal^−/−:Soat2^−/− mice was also much less than in their Lal^−/−:Soat2^+/+ littermates. In addition, there was a >70% reduction in plasma transaminase activities in the Lal^−/−:Soat2^−/− mice. These studies illustrate how the severity of disease in a mouse model for CESD can be substantially ameliorated by elimination of SOAT2 function.     

Role of the Dinaric Karst (western Balkans) in shaping the phylogeographic structure of the threatened crayfish Austropotamobius torrentium

1. This study examines phylogeography and phylogeny of the threatened stone crayfish, Austropotamobius torrentium, in order to elucidate the role of the Dinaric Karst geology in shaping the evolutionary history and genetic diversity of aquatic fauna in the western Balkans. Mitochondrial 16S rRNA and COI genes were partially sequenced from 188 and 159 crayfish, respectively, sampled from 70 localities. Phylogenetic relationships were reconstructed using four methods of phylogenetic inference. Divergence times between phylogroups were estimated in a Bayesian framework, and their demographic history was examined using neutrality tests and mismatch distribution analysis. 2. Seven geographically localised phylogroups separated by pronounced genetic gaps were found. Five of them have a distribution range within the northern‐central Dinaric (NCD) region, while the remaining two include populations from the southern Balkans (SB) and central and south‐eastern Europe (CSE). The oldest divergence event separated two NCD lineages from the rest of populations in the Late Miocene or Early Pliocene. Divergences amongst the five NCD phylogroups and SB + CSE occurred in the Pliocene. The most recent split separated SB and CSE phylogroups during the Late Pliocene. For both genes, uncorrected pairwise divergences between most of the phylogroups (4.1–8.7% for COI and 1.6–4.8% for 16S rRNA) were of the same range as, or higher than, some of the interspecific distances previously reported for the genus Austropotamobius. 3. Geographically isolated and deeply divergent cryptic monophyletic phylogroups within A. torrentium in the NCD region arose in the course of intensification of Neotectonic movements during the Pliocene and the beginning of the Pleistocene and the development of karstification that has heavily fragmented the palaeohydrography of the area. The results confirm a gradual north–south expansion of stone crayfish during the pre‐Pleistocene that preceded the rapid northward post‐glacial re/colonisation of central Europe (CSE phylogroup) through the Danube drainage. 4. Austropotamobius torrentium comprises morphologically cryptic but molecularly distinct taxa. Considering the relatively small geographical areas they inhabit, the NCD phylogroups of stone crayfish should be given the highest conservation priority.     

The phylogeny of the hominoid primates: a statistical analysis of the DNA-DNA hybridization data

Sibley and Ahlquist compared the single-copy nuclear DNA sequences of the hominoid primates using DNA-DNA hybridization. From this data set they estimated a phylogeny that clusters man and chimpanzees using a distance Wagner procedure. However, no assessment of statistical confidence in this estimated phylogeny was made, despite the fact that their data set contains internal inconsistencies concerning the correct branching order. This paper presents a modification of Pielou's Q- statistic that allows one to make nonparametric tests of phylogenetic relationship from distance data. The results of this analysis indicate that the estimated phylogeny of Sibley and Ahlquist is without statistical significance owing to the internal inconsistencies of the data set. A survey and additional analyses of other types of molecular data indicate that the phylogeny that clusters chimpanzees and gorillas and has the human lineage splitting off earlier is statistically consistent with all the molecular data (including the DNA-DNA hybridization data), whereas the phylogeny estimated by Sibley and Ahlquist can be rejected at the 5% level using the data on restriction- endonuclease sites in the mitochondrial genome.      

Water loss from Jackass Penguin Spheniscus demersus eggs during natural incubation

The water budget of incubating Jackass Penguin eggs was studied on Marcus Island, South Africa, and complementary measurements were made in the laboratory. The mean ambient temperature was 16-5 "C and the mean humidity was 12-4 Torr (89% relative humidity). The temperature of incubated live and water-filled eggs ranged between 14^oCand 37 ^oC. The mean calculated egg temperature was 34-9' C. The mean brood patch temperature was 37-1 ^oC, slightly lower than the cloacal temperature (37.8 ^oC). The mean brood patch area was about 38 cm². The rate of water loss was 411 mg day^-1. The total diffusive water loss during 37 days of incubation was, as predicted, 15-2% of the initial 100-3 g egg mass. The total pore number was 6245 per egg and the shell thickness was 577 fira. It is suggested that the eggshell parameters, incubation length and nesting behaviour are compensated in such a way that an egg-to-nest water vapour pressure difference lower than commonly found is sufficient to bring about the normal total water loss.