Oligonucleotide-assisted cleavage and ligation: a novel directional DNA cloning technology to capture cDNAs. Application in the construction of a human immune antibody phage-display library

The use of oligonucleotide-assisted cleavage and ligation (ONCL), a novel approach to the capture of gene repertoires, in the construction of a phage-display immune antibody library is described. ONCL begins with rapid amplification of cDNA ends to amplify all members equally. A single, specific cut near 5′ and/or 3′ end of each gene fragment (in single stranded form) is facilitated by hybridization with an appropriate oligonucleotide adapter. Directional cloning of targeted DNA is accomplished by ligation of a partially duplex DNA molecule (containing suitable restriction sites) and amplification with primers in constant regions. To demonstrate utility and reliability of ONCL, a human antibody repertoire was cloned from IgG mRNA extracted from human B-lymphocytes engrafted in Trimera mice. These mice were transplanted with peripheral blood lymphocytes from Candida albicans infected individuals and subsequently immunized with C.albicans glyceraldehyde-3-phosphate dehydrogenase (GAPDH). DNA sequencing showed that ONCL resulted in efficient capture of gene repertoires. Indeed, full representation of all V_H families/segments was observed showing that ONCL did not introduce cloning biases for or against any V_H family. We validated the efficiency of ONCL by creating a functional Fab phage-display library with a size of 3.3 × 10¹⁰ and by selecting five unique Fabs against GAPDH antigen.     

The comparative method rules! Codon volatility cannot detect positive Darwinian selection using a single genome sequence

All established methods for detecting positive selection at the molecular level rely on comparisons between nucleotide sequences. An exceptional method that purports to detect selection on the basis of a single genomic sequence has recently been proposed. This method uses a measure called "codon volatility," defined for each codon as the ratio between the number of nonsynonymous codons that differ from the codon under study at a single nucleotide position and the number of sense codons that differ from the codon under study at a single nucleotide position. Here, we examine various properties of codon volatility and its derivatives and use simulation of evolutionary processes to determine whether they can be used to detect selective pressures. Codons for only four amino acids (glycine, leucine, arginine, and serine) show any variation in codon volatility. Thus, codon volatility is mainly a proxy for amino acid usage, rather than for codon usage, with 65% of all synonymous changes and 27% of all nonsynonymous changes being undetectable by this measure. Genes identified by the volatility method as being subject to positive selection tend to have idiosyncratic amino acid compositions (e.g., they are glycine rich or arginine poor). An additional property of codon volatility is the near zero variance of its mean expectation, which translates into overestimated statistical significance estimates, especially in the absence of corrections for multiple comparisons. A comparison with measures of selection inferred through comparative methodology reveals no relationship between the results of the two methods. Finally, we show that codon volatility can increase in the absence of positive Darwinian selection; that is, increased codon volatility is not indicative of positive selection.     

Genomic analysis of SARS-CoV-2 reveals local viral evolution in Ghana

The confirmed case fatality rate for the coronavirus disease 2019 (COVID-19) in Ghana has dropped from a peak of 2% in March to be consistently below 1% since May 2020. Globally, case fatality rates have been linked to the strains/clades of circulating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) within a specific country. Here we present 46 whole genomes of SARS-CoV-2 circulating in Ghana, from two separate sequencing batches: 15 isolates from the early epidemic (March 12–April 1 2020) and 31 from later time-points ( 25–27 May 2020). Sequencing was carried out on an Illumina MiSeq system following an amplicon-based enrichment for SARS-CoV-2 cDNA. After genome assembly and quality control processes, phylogenetic analysis showed that the first batch of 15 genomes clustered into five clades: 19A, 19B, 20A, 20B, and 20C, whereas the second batch of 31 genomes clustered to only three clades 19B, 20A, and 20B. The imported cases (6/46) mapped to circulating viruses in their countries of origin, namely, India, Hungary, Norway, the United Kingdom, and the United States of America. All genomes mapped to the original Wuhan strain with high similarity (99.5–99.8%). All imported strains mapped to the European superclade A, whereas 5/9 locally infected individuals harbored the B4 clade, from the East Asian superclade B. Ghana appears to have 19B and 20B as the two largest circulating clades based on our sequence analyses. In line with global reports, the D614G linked viruses seem to be predominating. Comparison of Ghanaian SARS-CoV-2 genomes with global genomes indicates that Ghanaian strains have not diverged significantly from circulating strains commonly imported into Africa. The low level of diversity in our genomes may indicate lower levels of transmission, even for D614G viruses, which is consistent with the relatively low levels of infection reported in Ghana.     

Characterization of Growth-Retardant Effects on Vegetative Growth of Date Palm Seedlings

The leaves, inflorescences, and fruit bunches of date palm trees develop at the crown, which is the apex of a single trunk that can be up to 25 m tall. Because palm trees are monocots, the leaves and inflorescences are generated deep within the trunk from a single meristem—the palm heart—and are inaccessible for direct bioregulator treatments. We followed the effects of two growth regulators, paclobutrazol and uniconazole-P, on the vegetative growth of young date palm seedlings. Both growth regulators constrained the elongation rate of the leaves and reduced the leaf length. Microscopic analysis of dissected palm hearts revealed that the growth retardants did not affect the generation of new leaves. However, the treatments did change the dimensions of the developing embryonic leaves as well as the shape of the apical meristem. The results suggest the possible future use of growth retardants to reduce trunk height, enabling cheaper, safer, and more efficient management of date palm trees.     

Cognitive Performance during Sustained Wakefulness: A Low Dose of Caffeine Is Equally Effective as Modafinil in Alleviating the Nocturnal Decline

Cognitive performance at night exhibits a substantial drop, typically before dawn. One of the means of dealing with this phenomenon, as well as with the accompanying sleepiness during sustained wakefulness, is the administration of stimulants. The most widely used and well‐documented stimulants are caffeine, amphetamines, and modafinil. Of these, amphetamines are the least recommended, as they may severely affect behavior. Caffeine and modafinil seem to produce relatively milder side effects and usually only at high doses. Previous comparison studies have revealed equal efficacy of both the stimulants in maintaining alertness and performance during sustained wakefulness. However, these studies used relatively high, and thus not completely safe, doses of these drugs (600 mg caffeine and 400 mg modafinil). Therefore, the aim of the present study was to assess the efficacy of a low and medically safe dose of caffeine (200 mg) and modafinil (200 mg) in maintaining cognitive performance during sustained wakefulness. A flight simulation task was chosen for the assessment of the stimulants in a counter‐balanced, within‐subject design under four different conditions: baseline (no drugs), placebo, caffeine (200 mg), and modafinil (200 mg). The equal effectiveness of both drugs in abolishing the nocturnal drop in cognitive performance, as well as of oral temperature and blood pressure, supported the use of low doses of caffeine and modafinil for the maintenance of alertness in healthy subjects during sustained wakefulness.     

Quercetin alleviated H2O2‐induced apoptosis and steroidogenic impairment in goat luteinized granulosa cells

Quercetin (Que) is a natural flavonoid in most plants. Luteinized granulosa cell (LGC) culture is necessary for the study of follicle growth/differentiation. In the present study, we analyzed the role of Que in steroid production and apoptosis in hydrogen peroxide (H₂O₂)‐treated goat LGCs. The results showed that treatment with H₂O₂ induced apoptosis in goat LGCs, and treatment with Que decreased LGC apoptosis induced by H₂O₂ (P < .05), accompanied with the different expressions of BAX, BCL‐2, Caspase 3, and Cleaved caspase 3. Meanwhile, the messenger RNA expressions of nuclear factor erythroid 2 like 2 (Nrf2) and its downstream genes were upregulated with H₂O₂ +Que treatment, accompanied by the increased cellular viability (P < .05). Furthermore, Que alleviated H₂O₂‐induced reduction in the secretion of progesterone (P₄) (P < .05); however, it had no effect on the secretion of estrogen (E₂). Simultaneously, the expressions of StAR and P450scc were increased when treated with Que +H₂O₂, compared with the group treated with only H₂O₂ (P < .05). In conclusion, it is observed that Que could alleviate the H₂O₂‐induced apoptosis and steroidogenic impairment in goat LGCs, which might be mediated by the Nrf2 pathway.     

Networks uncover hidden lexical borrowing in Indo-European language evolution

Language evolution is traditionally described in terms of family trees with ancestral languages splitting into descendent languages. However, it has long been recognized that language evolution also entails horizontal components, most commonly through lexical borrowing. For example, the English language was heavily influenced by Old Norse and Old French; eight per cent of its basic vocabulary is borrowed. Borrowing is a distinctly non-tree-like process--akin to horizontal gene transfer in genome evolution--that cannot be recovered by phylogenetic trees. Here, we infer the frequency of hidden borrowing among 2346 cognates (etymologically related words) of basic vocabulary distributed across 84 Indo-European languages. The dataset includes 124 (5%) known borrowings. Applying the uniformitarian principle to inventory dynamics in past and present basic vocabularies, we find that 1373 (61%) of the cognates have been affected by borrowing during their history. Our approach correctly identified 117 (94%) known borrowings. Reconstructed phylogenetic networks that capture both vertical and horizontal components of evolutionary history reveal that, on average, eight per cent of the words of basic vocabulary in each Indo-European language were involved in borrowing during evolution. Basic vocabulary is often assumed to be relatively resistant to borrowing. Our results indicate that the impact of borrowing is far more widespread than previously thought.     

Requirement for CD4 T cell help in maintenance of memory CD8 T cell responses is epitope dependent

CD4 Th cells play critical roles in stimulating Ab production and in generating primary or maintaining memory CTL. The requirement for CD4 help in generating and maintaining CTL responses has been reported to vary depending on the vector or method used for immunization. In this study, we examined the requirement for CD4 T cell help in generating and maintaining CTL responses to an experimental AIDS vaccine vector based on live recombinant vesicular stomatitis virus (VSV) expressing HIV Env protein. We found that primary CD8 T cell responses and short-term memory to HIV Env and VSV nucleocapsid (VSV N) proteins were largely intact in CD4 T cell-deficient mice. These responses were efficiently recalled at 30 days postinfection by boosting with vaccinia recombinants expressing HIV Env or VSV N. However, by 60 days postinfection, the memory/recall response to VSV N was lost in CD4-deficient mice, while the recall response HIV Env was partially maintained in the same animals for at least 90 days. This result indicates that there are epitope-specific requirements for CD4 help in the maintenance of memory CD8 T cell responses. Our results also suggest that choice of epitopes might be critical in an AIDS vaccine designed to protect against disease in the context of reduced or declining CD4 T cell help.     

Prenatal programming of rat proximal tubule Na+/H+ exchanger by dexamethasone

Prenatal administration of dexamethasone causes hypertension in rats when they are studied as adults. Although an increase in tubular sodium reabsorption has been postulated to be a factor programming hypertension, this has never been directly demonstrated. The purpose of this study was to examine whether prenatal programming by dexamethasone affected postnatal proximal tubular transport. Pregnant Sprague-Dawley rats were injected with intraperitoneal dexamethasone (0.2 mg/kg) daily for 4 days between the 15th and 18th days of gestation. Prenatal dexamethasone resulted in an elevation in systolic blood pressure when the rats were studied at 7-8 wk of age compared with vehicle-treated controls: 131 +/- 3 vs. 115 +/- 3 mmHg (P < 0.001). The rate of proximal convoluted tubule volume absorption, measured using in vitro microperfusion, was 0.61 + 0.07 nl.mm(-1).min(-1) in control rats and 0.93+ 0.07 nl.mm(-1).min(-1) in rats that received prenatal dexamethasone (P < 0.05). Na(+)/H(+) exchanger activity measured in perfused tubules in vitro using the pH-sensitive dye BCECF showed a similar 50% increase in activity in proximal convoluted tubules from rats treated with prenatal dexamethasone. Although there was no change in abundance of NHE3 mRNA, the predominant luminal proximal tubule Na(+)/H(+) exchanger, there was an increase in NHE3 protein abundance on brush-border membrane vesicles in 7- to 8-wk-old rats receiving prenatal dexamethasone. In conclusion, prenatal administration of dexamethasone in rats increases proximal tubule transport when rats are studied at 7-8 wk old, in part by stimulating Na(+)/H(+) exchanger activity. The increase in proximal tubule transport may be a factor mediating the hypertension by prenatal programming with dexamethasone.