HLA and AIDS: a cautionary tale

The human major histocompatibility complex HLA has been implicated repeatedly as a regulator of the outcome of HIV exposure and infection. A new study of long-term survivors who naturally depress HIV-1 replication and avoid the signs of AIDS for years after infection suggests that homozygosity for a group of HLA-B locus alleles termed Bw4 confers resistance, ostensibly by regulating natural killer cell-ligand interactions. However, close inspection of the accumulated evidence raises some questions and urges validation of the potential Bw4 effects in additional studies.     

Laboratory-dependent bacterial ecology: a cautionary tale

Although laboratory dependence is an acknowledged problem in microbiology, it is seldom intensively studied or discussed. We demonstrate that laboratory dependence is real and quantifiable even in the popular model Escherichia coli. Here laboratory effects alter the equilibrium composition of a simple community composed of two strains of E. coli. Our data rule out changes in the bacterial strains, chemical batches, and human handling but implicate differences in growth medium, especially the water component.     

Naming taxa from cladograms: a cautionary tale

The recent publication of a new hypothesis of cladistic relationships among American frogs referred to the genus Rana, accompanied by a new taxonomy and a new nomenclature of this group [Hillis D.M., Wilcox, T.P., 2005. Phylogeny of the New World true frogs (Rana). Molecular Phylogenetics and Evolution 34, 299-314], draws attention to the problems posed by the use of a "double nomenclature", following both the rules of the International Code of Zoological Nomenclature (designated here as "onomatophore-based nomenclature") and the rules of the draft Phylocode (designated here as "definition-based nomenclature"). These two nomenclatural systems, which rely upon widely different theoretical bases, are incompatible, and the latter cannot be viewed as a "modification" of the former. Accordingly, scientific names (nomina) following both systems should be clearly distinguished in scientific publications. Onomatophore-based nomina should continue to be written as they have been for about 250 years, whereas definition-based nomina should be written in a specific way, e.g., Lithobates. The combined use of both nomenclatural systems for the same taxonomy in the same paper requires good knowledge and careful respect of the rules of the Code regarding availability, allocation and validity of nomina. As shown by this example, not doing so may result in various problems, in particular in publishing nomina nuda or in using nomenclatural ranks invalid under the current Code. Attention is drawn to the fact that new nomina published without diagnostic characters are not available under the Code, and that the latter currently forbids the use of more than two ranks (subgenus and "aggregate of species") between the ranks genus and species.     

FURIN and placental syncytialisation: a cautionary tale

FURIN is a pro-protein convertase previously shown to be important for placental syncytialisation (Zhou et al. [1]), a process of cell fusion whereby placental cytotrophoblast cells fuse to form a multinucleated syncytium. This finding has been broadly accepted however, we have evidence suggesting the contrary. Spontaneously syncytialising term primary human trophoblast cells and BeWo choriocarcinoma cells were treated with either FURIN siRNA or negative control siRNA or the protease inhibitor, DEC-RVKR-CMK, or vehicle. Cells were then left to either spontaneously syncytialise (primary trophoblasts) or were induced to syncytialise with forskolin (BeWo). Effects on syncytialisation were measured by determining human chorionic gonadotrophin secretion and E-cadherin protein levels. We showed that FURIN is not important for syncytialisation in either cell type. However, in primary trophoblasts another protease also inhibited by DEC-RVKR-CMK, may be involved. Our results directly contrast with those published by Zhou et al. Zhou et al. however, used first trimester villous explants to study syncytialisation, and we used term primary trophoblasts. Therefore, we suggest that FURIN may be involved in syncytialisation of first trimester trophoblasts, but not term trophoblasts. What is more concerning is that our results using BeWo cells do not agree with their results, even though for the most part, we used the same experimental design. It is unclear why these experiments yielded different results, however we wanted to draw attention to simple differences in measuring syncytialisation or flaws in method reporting (including omission of cell line source and passage numbers, siRNA concentration and protein molecular weights) and choice of immunoblot loading controls, that could impact on experimental outcomes. Our study shows that careful reporting of methods by authors and thorough scrutiny by referees are vital. Furthermore, a universal benchmark for measuring syncytialisation is required so that various studies of syncytialisation can be validated.Subject terms: Proteases, Differentiation     

Dangerous weapons: a cautionary tale of CRISPR defence

The Cas/CRISPR system of prokaryotes is a recently recognized defence system that protects cells from invasion by foreign nucleic acids, such as plasmids or bacteriophages. Using the archaeon Sulfolobus, Gudbergsdottir et al. have demonstrated that when foreign DNA is both a target of the CRISPR system and also carries a gene that is essential for cell growth, surviving cells display mutations in the CRISPR system that eliminate the target directed spacer. This simulates natural scenarios where the invading DNA cannot be readily destroyed, such as a temperate bacteriophage that manages to evade CRISPR attack and integrate into the genome, so becoming ‘self’. CRISPR deletions ranged from precise removal of a single spacer up to elimination of an entire CRISPR array but the mechanism involved is not understood. Another surprising feature was that from one to two base mutations in the same target protospacer did not affect recognition by the Sulfolobus CRISPR system, something unexpected from studies in bacteria where spacer and target protospacer must match exactly. This study shows that even though the CRISPR system is a powerful weapon it can also be a dangerous one for the cell that carries it.     

Tris-borate is a poor counterion for RNA: a cautionary tale for RNA folding studies

Native polyacrylamide gel electrophoresis is a powerful approach for visualizing RNA folding states and folding intermediates. Tris-borate has a high-buffering capacity and is therefore widely used in electrophoresis-based investigations of RNA structure and folding. However, the effectiveness of Tris-borate as a counterion for RNA has not been systematically investigated. In a recirculated Hepes/KCl buffer, the catalytic core of the bI5 group I intron RNA undergoes a conformational collapse characterized by a bulk transition midpoint, or Mg_1/2, of ~3 mM, consistent with extensive independent biochemical experiments. In contrast, in Tris-borate, RNA collapse has a much smaller apparent Mg_1/2, equal to 0.1 mM, because in this buffer the RNA undergoes a different, large amplitude, folding transition at low Mg²⁺ concentrations. Analysis of structural neighbors using a short-lived, RNA-tethered, photocrosslinker indicates that the global RNA structure eventually converges in the two buffer systems, as the divalent ion concentration approaches ~1 mM Mg²⁺. The weak capacity of Tris-borate to stabilize RNA folding may reflect relatively unfavorable interactions between the bulky Tris-borate ion and RNA or partial coordination of RNA functional groups by borate. Under some conditions, Tris-borate is a poor counterion for RNA and its use merits careful evaluation in RNA folding studies.     

Inhibitors of superoxide dismutases: A cautionary tale

An extensive search resulted in the identification of pamoic acid as an inhibitor of superoxide dismutases. Pamoic acid appeared to rapidly and reversibly inhibit all types of superoxide dismutases and did so in both the cytochrome c reduction and in the dianisidine photooxidation assays, used to measure this activity. It could nevertheless be shown that pamoic acid did not at all inhibit superoxide dismutase but rather diminished the sensitivity of the assays. The mechanism proposed to account for this effect involved oxidation of pamoate, by O₂^?, to yield a pamoate radical which can then reduce cytochrome c or oxidize pyrogallol. Pamoate thus competes with superoxide dismutase for the available O₂^?, without affecting the observable effects of that O₂^? upon cytochrome c or upon pyrogallol. It consequently makes these assays less responsive to superoxide dismutase, while appearing to be without effect in the absence of superoxide dismutase. Several of the predicted consequences of this proposal were affirmed. Other workers, interested in finding inhibitors for superoxide dismutases, are hereby forwarned of this subtle snare.     

Bateman gradients in field and laboratory studies: a cautionary tale

Since tools of molecular genetics became readily available,our understanding of bird mating systems has undergone a revolution.The majority of passerine species investigated are sociallymonogamous, but have been shown to be genetically polygamous.Data sets from natural populations of juncos suggest that multiplemating by females results in a sexual selection gradient assteep for females as for males (a result that does not supportBateman's predictions). However, in males, fitness is enhanceddirectly through fertilization success with multiple matings;in females fitness benefits may be enhanced immediately throughdirect access to food, protection against predators, or otherresources received from males, or they may be delayed throughimprovement in offspring quality (e.g., through good genes,or greater genetic compatibility between the female and theextra-pair male). But a steep sexual selection gradient forfemales can be difficult to interpret. If all females copulatewith multiple partners that are equally likely to fertilizeeggs, then females that produce larger clutch sizes, for anyreason, will appear to have copulated with more males. Thatis, multiple sires have a higher probability of detection inlarger clutches than in smaller ones, giving the impressionthat females that mate with multiple males increase their reproductivesuccess. Yet, in most studies in which there is a correlationbetween number of offspring produced by females and number ofextra-pair males, causation has not been clearly establishedand other factors may explain the results. Additional complicationsin understanding male and female reproductive strategies are:(1) Molecular studies cannot detect extra-pair copulations thatdid not result in fertilizations; yet if a female acquires foodor other resources from extra-pair males, such extra-pair matingsmay have significant effects on female fitness. Thus, molecularstudies provide only a conservative estimate of the number ofextra-pair copulations or "mates" that a female has. (2) Clutchsize affects the probability that any given male will be successfulin fertilizing a female's eggs. Specifically, at any given point,a male's chances of fertilizing at least one egg in the female'sclutch will be greater as clutch size increases. We predictthat in avian species with small clutch sizes, males may beselected to be choosy and avoid extra-pair copulations, whilefemales should be selected to be less discriminating. Moreover,if extra-pair males provide resources that increase female fitness,the females should seek extra-pair copulations, whether or notthe males are likely to fertilize any of her eggs. Laboratory studies with insects have yielded clearer evidenceof the causal relationship between multiple mating and increasedfemale fitness. We review studies on a tenebrionid beetle inwhich female fecundity increases directly with number of mates.In these experiments, the nutritive value of the spermatophoresdoes not fully explain the increase in female reproductive success.     

Behavioral phenotyping of transgenic and knockout animals: a cautionary tale

Knockout and transgenic mice are extremely useful for behavioral research, especially for linking specific genes with behaviors. The authors present caveats to be aware of when using such mice in research situations.