A genetic system for direct selection of gene-positive clones during recombinational cloning in yeast

Transformation-associated recombination (TAR) is a cloning technique that allows specific chromosomal regions or genes to be isolated directly from genomic DNA without prior construction of a genomic library. This technique involves homologous recombination during spheroplast transformation between genomic DNA and a TAR vector that has 5′ and 3′ gene targeting sequences (hooks). Typically, TAR cloning produces positive YAC recombinants at a frequency of ~0.5%; the positive clones are identified by PCR or colony hybridization. This paper describes a novel TAR cloning procedure that selects positive clones by positive and negative genetic selection. This system utilizes a TAR vector with two targeting hooks, HIS3 as a positive selectable marker, URA3 as a negative selectable marker and a gene-specific sequence called a loop sequence. The loop sequence lies distal to a targeting hook sequence in the chromosomal target, but proximal to the targeting hook and URA3 in the TAR vector. When this vector recombines with chromosomal DNA at the gene-specific targeting hook, the recombinant YAC product carries two copies of the loop sequence, therefore, the URA3 negative selectable marker becomes mitotically unstable and is lost at high frequency by direct repeat recombination involving the loop sequence. Positive clones are identified by selecting against URA3. This method produces positive YAC recombinants at a frequency of ~40%. This novel TAR cloning method provides a powerful tool for structural and functional analysis of complex genomes.     

Biases associated with the use of underwater visual census techniques to quantify the density and size-structure of fish populations

Estimates of reef fish densities made by divers visually censusing 5 m wide strip transects were compared with capture-resight estimates calculated independently using data on the resighting frequencies of fish marked with colour-coded tags. The difference in density estimates between methods varied between species but with patterns consistent at the three eastern Tasmanian sites studied. Densities of the two most abundant species, the wrasses Notolabrus tetricus and Notolabrus fucicola, showed good agreement between methods. By contrast, populations of the two monacanthids Meuschenia australis and Meuschenia freycineti were underestimated by an order of magnitude in strip transects relative to capture-resight, while populations of the open-water latrid Latridopsis forsteri were overestimated.For all common fish species observed in strip transects, variation in density estimates between divers was extremely low compared to variation between sites and between months. Variation in density estimates between different days within a week and between 50 m blocks located 50 m apart was also negligible; however, residual error associated with variation by a single diver within a block and day was high. Biases associated with changing detectability of fishes in open versus densely vegetated habitats were assessed by algal removal experiments, and were found to be negligible or non-significantly low for five of the six species examined. The exceptional species—N. tetricus—exhibited a 70% rise in fish sighted by diver following algal clearance despite trapping data indicating no increase in fish numbers.Diver estimates of fish length were on average 7% greater than measured lengths. Divers possessed a clear tendency to make increasingly inaccurate size estimates as fish length deviated in either direction from 300 mm. Lengths of 175-mm animals were underestimated by ≈20% and 400-mm fishes were overestimated by ≈10%. These changes in diver bias with fish size were largely independent of fish species, site and diver. The precision of size estimates, as indicated by the standard deviation of bias, also varied with fish size, with values varying from ≈13% at 200 mm fish length to ≈8% at 400 mm length. The decline in precision at small body size largely reflected size intervals used by divers to bin data. Divers appear capable of making more precise size estimates than the 25-mm interval used at small fish sizes.     

Inhibition of a dual chamber pacemaker by oversensing of far-field atrial depolarisation in a patient after His-bundle ablation

After His-bundle ablation, a 54-year-old pacemaker-dependent patient suffered from severe presyncopal attacks one year after pacemaker replacement. The attacks were caused by ventricular inhibition due to atrial far-field sensing during paroxysmal atrial flutter.     

White-faced capuchin monkeys show triadic awareness in their choice of allies

The social intelligence hypothesis, which holds that social challenges have selected for increased intelligence and social skills, has been supported by evidence that, in catarrhine primates, individuals know about the characteristics of groupmates' social relationships. Evidence for such ‘triadic awareness’ has not been sought for platyrrhine primates, although two platyrrhine genera, capuchins, Cebus, and squirrel monkeys, Saimiri, are among the most highly encephalized primates. We examined patterns of coalitionary recruitment in wild white-faced capuchins, C. capucinus. Analyses have shown that more dominant individuals are more likely to join aggressive coalitions than low-rankers, and that individuals preferentially support those with whom they have stronger affiliative relationships. Data from 110 fights, analysed using simulation techniques that produced distributions of results expected under null hypotheses, revealed that contestants preferentially solicited prospective coalition partners that (1) were dominant to their opponents, and (2) had better social relationships (higher ratios of affiliative/cooperative interactions to agonistic interactions) with themselves than with their opponents. Further analyses showed that soliciting dominant partners could be explained by either of two simpler rules, ‘Solicit an ally that outranks yourself’ or ‘Solicit the highest-ranking available individual’. However, soliciting partners with better social relationships appears to indicate triadic awareness, because subjects did not preferentially solicit the nearby individual with whom they shared the highest-quality social relationship. Effects of relative relationship quality on coalition solicitation decisions were independent of effects of dominance rank.     

Phylogenetic and Evolutionary Relationships among Yellow Fever Virus Isolates in Africa

Previous studies with a limited number of strains have indicated that there are two genotypes of yellow fever (YF) virus in Africa, one in west Africa and the other in east and central Africa. We have examined the prM/M and a portion of the E protein for a panel of 38 wild strains of YF virus from Africa representing different countries and times of isolation. Examination of the strains revealed a more complex genetic relationship than previously reported. Overall, nucleotide substitutions varied from 0 to 25.8% and amino acid substitutions varied from 0 to 9.1%. Phylogenetic analysis using parsimony and neighbor-joining algorithms identified five distinct genotypes: central/east Africa, east Africa, Angola, west Africa I, and west Africa II. Extensive variation within genotypes was observed. Members of west African genotype II and central/east African genotype differed by 2.8% or less, while west Africa genotype I varied up to 6.8% at the nucleotide level. We speculate that the former two genotypes exist in enzootic transmission cycles, while the latter is genetically more heterogeneous due to regular human epidemics. The nucleotide sequence of the Angola genotype diverged from the others by 15.7 to 23.0% but only 0.4 to 5.6% at the amino acid level, suggesting that this genotype most likely diverged from a progenitor YF virus in east/central Africa many years ago, prior to the separation of the other east/central African strains analyzed in this study, and has evolved independently. These data demonstrate that there are multiple genotypes of YF virus in Africa and suggest independent evolution of YF virus in different areas of Africa.     

Genbank accession #: AF 124983

Plant Molecular Biology -     

The MKK7 Gene Encodes a Group of c-Jun NH2-Terminal Kinase Kinases

The c-Jun NH₂-terminal protein kinase (JNK) is a member of the mitogen-activated protein kinase (MAPK) group and is an essential component of a signaling cascade that is activated by exposure of cells to environmental stress. JNK activation is regulated by phosphorylation on both Thr and Tyr residues by a dual-specificity MAPK kinase (MAPKK). Two MAPKKs, MKK4 and MKK7, have been identified as JNK activators. Genetic studies demonstrate that MKK4 and MKK7 serve nonredundant functions as activators of JNK in vivo. We report here the molecular cloning of the gene that encodes MKK7 and demonstrate that six isoforms are created by alternative splicing to generate a group of protein kinases with three different NH₂ termini (α, β, and γ isoforms) and two different COOH termini (1 and 2 isoforms). The MKK7α isoforms lack an NH₂-terminal extension that is present in the other MKK7 isoforms. This NH₂-terminal extension binds directly to the MKK7 substrate JNK. Comparison of the activities of the MKK7 isoforms demonstrates that the MKK7α isoforms exhibit lower activity, but a higher level of inducible fold activation, than the corresponding MKK7β and MKK7γ isoforms. Immunofluorescence analysis demonstrates that these MKK7 isoforms are detected in both cytoplasmic and nuclear compartments of cultured cells. The presence of MKK7 in the nucleus was not, however, required for JNK activation in vivo. These data establish that the MKK4 and MKK7 genes encode a group of protein kinases with different biochemical properties that mediate activation of JNK in response to extracellular stimuli.     

Changes in the associations of race and rurality with SARS-CoV-2 infection,mortality, and case fatality in the United States from February 2020 to March 2021: A population-based cohort study

BackgroundWe examined whether key sociodemographic and clinical risk factors for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection and mortality changed over time in a population-based cohort study.Methods and findingsIn a cohort of 9,127,673 persons enrolled in the United States Veterans Affairs (VA) healthcare system, we evaluated the independent associations of sociodemographic and clinical characteristics with SARS-CoV-2 infection (n = 216,046), SARS-CoV-2–related mortality (n = 10,230), and case fatality at monthly intervals between February 1, 2020 and March 31, 2021. VA enrollees had a mean age of 61 years (SD 17.7) and were predominantly male (90.9%) and White (64.5%), with 14.6% of Black race and 6.3% of Hispanic ethnicity. Black (versus White) race was strongly associated with SARS-CoV-2 infection (adjusted odds ratio [AOR] 5.10, [95% CI 4.65 to 5.59], p-value <0.001), mortality (AOR 3.85 [95% CI 3.30 to 4.50], p-value < 0.001), and case fatality (AOR 2.56, 95% CI 2.23 to 2.93, p-value < 0.001) in February to March 2020, but these associations were attenuated and not statistically significant by November 2020 for infection (AOR 1.03 [95% CI 1.00 to 1.07] p-value = 0.05) and mortality (AOR 1.08 [95% CI 0.96 to 1.20], p-value = 0.21) and were reversed for case fatality (AOR 0.86, 95% CI 0.78 to 0.95, p-value = 0.005). American Indian/Alaska Native (AI/AN versus White) race was associated with higher risk of SARS-CoV-2 infection in April and May 2020; this association declined over time and reversed by March 2021 (AOR 0.66 [95% CI 0.51 to 0.85] p-value = 0.004). Hispanic (versus non-Hispanic) ethnicity was associated with higher risk of SARS-CoV-2 infection and mortality during almost every time period, with no evidence of attenuation over time. Urban (versus rural) residence was associated with higher risk of infection (AOR 2.02, [95% CI 1.83 to 2.22], p-value < 0.001), mortality (AOR 2.48 [95% CI 2.08 to 2.96], p-value < 0.001), and case fatality (AOR 2.24, 95% CI 1.93 to 2.60, p-value < 0.001) in February to April 2020, but these associations attenuated over time and reversed by September 2020 (AOR 0.85, 95% CI 0.81 to 0.89, p-value < 0.001 for infection, AOR 0.72, 95% CI 0.62 to 0.83, p-value < 0.001 for mortality and AOR 0.81, 95% CI 0.71 to 0.93, p-value = 0.006 for case fatality). Throughout the observation period, high comorbidity burden, younger age, and obesity were consistently associated with infection, while high comorbidity burden, older age, and male sex were consistently associated with mortality. Limitations of the study include that changes over time in the associations of some risk factors may be affected by changes in the likelihood of testing for SARS-CoV-2 according to those risk factors; also, study results apply directly to VA enrollees who are predominantly male and have comprehensive healthcare and need to be confirmed in other populations.ConclusionsIn this study, we found that strongly positive associations of Black and AI/AN (versus White) race and urban (versus rural) residence with SARS-CoV-2 infection, mortality, and case fatality observed early in the pandemic were ameliorated or reversed by March 2021.

George Ioannou and co-workers study the distribution of SARS-CoV-2 infections and outcomes among the United States population.     

Identification of natural compounds tubercidin and lycorine HCl against small‐cell lung cancer and BCAT1 as a therapeutic target

Although small‐cell lung cancer (SCLC) accounts for a small fraction of lung cancer cases (~15%), the prognosis of patients with SCLC is poor with an average overall survival period of a few months without treatment. Current treatments include standard chemotherapy, which has minimal efficacy and a newly developed immunotherapy that thus far, benefits a limited number of patients. In the current study, we screened a natural product library and identified 5 natural compounds, in particular tubercidin and lycorine HCl, that display prominent anti‐SCLC activities in vitro and in vivo. Subsequent RNA‐sequencing and functional validation assays revealed the anti‐SCLC mechanisms of these new compounds, and further identified new cellular factors such as BCAT1 as a potential therapeutic target with clinical implication in SCLC patients. Taken together, our study provides promising new directions for fighting this aggressive lung cancer.