Reprogrammed iPS-cells are not safe from self immune-attack.

The transition of Stem Cells (SC) from emergence to utter-prominence on the therapeutic-horizon has been phenomenal. While the promise of cell-based therapeutics has been there, applications of pluripotent SC’s for treatment of human patients have largely been under a cloud of controversy and doubt. While there are several, few doubts are glaring. One among those is the possible immunological-rejection of SC’s by the patient (since they are derived from other humans, grafted SC’s are viewed as allografts or non-self by the immune system). There were few attempts to overcome this impediment, not necessarily with the right moral-code, though (read Hwang et al., Science).

This virtual blind-end was resuscitated recently by the work of Yamanaka et al. and others, who found out a way to, well, at least theoretically, get around the immunological-rejection problem.

They made pluripotent cells from adult human skin, and named them induced-pluripotent-stem-cells (iPSCs). To cut a very-long story short, Yamanaka’s group dumped the original (and vastly controversial) method of pluripotent stem-cell derivation from human-embryos, and generated pluripotent cells from adult skin cells instead, by a method of cellular reprogramming (de-differentiation) [1]. Everybody rejoiced. This was no mean achievement, in terms of both the science behind it and the effort. This was the holy-grail of cell-based therapeutics, good news for both patients and the therapeutic-industry. This study was hailed, since it was reasoned that, at least theoretically, iPSCs by virtue of their origin from the patient’s own cells, would be recognized as ‘self’ and hence would not be mauled by the patient’s immune system, and hence could be a safe bet for cell-based therapies like cell-transfers, organ-grafts etc. This was followed by a series of iPS-cell derivations, from mice as well as humans [2,3,4].

Schematic representation of derivation of pluripotent iPS-cells derived from self-cells upon de-differentiation, and are capable of differentiation into all types of cells under in-vitro conditions or upon transplantation for therapeutic purposes. Since they are formed from self-cells, they were thought to be resistant to immunological attack, a belief proved wrong in this study.

All was fine, until this ‘letter’ in Nature spoiled the party. It was shown by a group from University of California (UC), San Diego (SD), that the underlying assumption that iPSCs would be immunologically tolerated was incorrect [5]. They showed that iPSCs, notwithstanding the reason of immune-tolerance, were actually actively repelled by the body’s defenses. Immunologist Yang Xu of UCSD and his colleagues tested what happened to several kinds of pluripotent cells when they were transplanted into genetically matched mice. Inbred mouse strains are the genetic equivalent of identical twins, and they can serve as organ donors for each other without any immune suppression. The researchers used two popular inbred strains, called B6 and 129, for their experiments.

Results:

When the researchers implanted ES cells from a B6 mouse embryo into a B6 mouse, it formed a typical growth, called a teratoma, which is a mixture of differentiating cell types (Teratoma formation is a standard test of pluripotency for ES and iPS cells). ES cells from a 129 mouse, on the other hand, were unable to form teratomas in B6 mice because the animals’ immune systems attacked the cells, which they recognized as foreign.

The researchers then implanted iPS cells made from B6 mouse cells into B6 mice. To their surprise, many of the cells failed to form teratomas at all-similar to what the researchers saw when they transplanted ES cells from one mouse strain to another.

The teratomas that did grow were soon attacked by the recipient’s immune system and were rejected, which is the same as that triggered by organ transplant between individuals.

The immune reaction was less severe when the researchers used iPS cells made with a newer technique. The new method ensures that the added genes that trigger reprogramming turn off after they’ve done their job. But the reaction didn’t go away completely. The researchers showed that the iPS cell teratomas expressed high levels of certain genes that could trigger immune cells to attack. That is probably due to incomplete reprogramming that leaves some genes misexpressed, Xu says.

Discussion: what next. 

> The results add to a series of findings that iPS cells differ in subtle but potentially important ways from ES cells [writeup here]. 

> The team used two different methods to make the iPS cells, and they showed slightly different propensities to trigger immune rejection, so it may be that reprogramming methods can be fine-tuned to avoid the problem altogether.

References:

[1] Takahashi, K. & Yamanaka, S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663–676 (2006).

[2] Takahashi, K. et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861–872 (2007).

[3] Yu, J. et al. Induced pluripotent stem cell lines derived from human somatic cells. Science 318, 1917–1920 (2007).

[4] Park, I.-H. et al. Reprogramming of human somatic cells to pluripotency with defined factors. Nature 451, 141–146 (2008).

[5] Tongbiao Zhao, Zhen-Ning Zhang, Zhili Rong & Yang Xu. Immunogenicity of induced pluripotent stem cells. Nature, doi:10.1038/nature10135

Evidence of a Pluripotent Human Embryonic Stem Cell Line Derived from a Cloned Blastocyst. Woo Suk Hwang et al. Science 2004:  303; 1669-1674

Algae could help clean-up nuclear-accident sites

              The 'Mushroom-cloud' formed due to a nuclear explosion (image adapted from)

The dark images and darker consequences of several nuclear disasters like the Chernobyl (April 26, 1986), Kyshtym (September 29, 1957), Three-Mile island (March 28, 1979) and the very-ongoing Japan’s Fukushima Nuclear accident constantly remind us of the Frankensteinian possibilities of nuclear-capability. With world governments heralding nuclear energy as a panacea for all energy needs, there has been a spurt in the proliferation of nuclear technology, capability and of course, nuclear reactors. And as the world clamours for a debate on 'responsible' nuclear usage and simultaneously goes 'nuclear' at a tumbling speed we remain naked under the threatening shroud of a looming nuclear disaster. 

While we recklessly roll towards reactors, our capabilities at cleaning-up a nuclear-spillage or tidying-up post radioactive-leakage remains abysmally poor. Our technology to efficiently dispose off radioactive garbage is juvenile at best, and Nature, again, seems to show the way.

from Manhattan to Moniliferum

A recent paper [1] by Minna krejci's group, a materials scientist at Northwestern University in Evanston Illinois, claims that the common freshwater alga Closterium moniliferum might hold the key to an efficient nuclear-clean-up-act, after humans have messed up! Members of the desmid order, they are unicellular eukaryotic fresh-water alga popularly known for their distinctive crescent shapes measuring ~260 micron in length.

                                             Clostridium moniliferum (source)

These crescent-shaped C. moniliferum have an unusual ability to remove strontium from water, depositing it in crystals that form in subcellular structures known as vacuoles — an ability that could include the radioactive isotope strontium-90. The unicellular desmid green algae are ubiquitous in fresh water habitats and robust in lab in-vitro cultures, and as such are particularly suitable as a model system for Sr/Ba biomineralization [2].

Why is Strontium harmful?

Strontium lies exactly below calcium in the periodic table, and shares similar chemical properties and atomic size with calcium, so biological processes can't easily separate the two elements. That makes strontium-90, which has a half-life of about 30 years [3] a particularly dangerous radio-isotope: it can infiltrate body fluids like milk, and body tissues like bone marrow, bones, blood and others, where the harmful radiation that it emits can eventually lead to metastasis and cancer. It must be recalled that it was primarily Strontium-90 which caused havoc during the Chernobyl disaster of 1986. Unfortunately, reactor waste and accidental spills can contain up to ten billion times more of the harmless calcium than the dangerous strontium, making it extremely difficult to selectively clean up the strontium without also having to dispose of the harmless calcium. 

other methods, which are largely inefficient:

And, in the case of 90Sr, even the most advanced ion-exchange materials find it challenging to efficiently separate out Ca2+, Sr2+, and Ba2+ owing to their chemical similarity [4]. While  phytoremediation approaches utilizing the accumulation of environmental contaminants by green plants are becoming increasingly popular, the effectiveness of such approaches for Sr-90 sequestration are drastically reduced in the presence of Ca2+, due to the indiscriminate transport of Ca2+, Sr2+, and Ba2+ exhibited by most organisms [5].

Why is this alga (Clostridium moniliferum) significant?

This humble fresh-water alga has no particular interest in strontium: it mostly cares for barium. But as strontium happens to be midway between calcium and barium in atomic-size and other properties, so any of it that happens to be around gets crystallized as well. Calcium, as it turns out, even being far more abundant than either of the other two elements, is different enough to barium that it gets left behind, and doesn’t crystallize. The result is a crystal that is chiefly composed of barium, but is heavily enriched in strontium and has no calcium. 

BaSO4 crystals in C. moniliferum. a) Confocal microscopy image showing the lobes of the two chloroplasts (red); cell membrane in green. b) DIC image of BaSO4 crystals (arrow) in the terminal vacuole. c) SEM image of rhombic (arrowhead) and hexagonal (arrow) crystals that remain after cells have been ashed.

[image from paper (1)]

How do they do that?

The mechanism of barium or strontium entry into the organism is not well studied, but it is known that sulphate-rich vacuoles of the alga greatly aid formation of the crystals. Since, barium and strontium have relatively low solubility in sulphate solutions; they easily precipitate out to form crystals of BaSO4 in the sulphate rich small terminal vacuoles at the tips of the crescent-shaped cells. Do the crystals serve any physiological function? It’s not known yet.

The possibilities

> Now that it’s known that the organism actively hunts for barium, it is perhaps possible to enhance the uptake of strontium by tailoring the amount of barium in the algae's environment. 
> It would then be possible to ‘seed’ a spill of radioactive material, with barium to encourage the algae to grab the strontium of the nuclear waste. 
> It might also be possible to improve the process by tinkering with sulphate levels in the environment, thereby changing the amount of sulphate in the vacuoles, but indeed it would depend on an understanding of how cells might respond to altered conditions.

Then what?

Once isolated by the algae, the strontium could be kept in high-level nuclear waste repositories, while the rest of the waste could go to a less expensive lower-level repository, saving space and money. In any case, the hundreds of millions of litres of stored strontium containing nuclear waste in the United States alone.

questions and curiosities

> It’s not yet tested how well the algae survive in the presence of radioactivity. But, since the process begins quickly, wherein cells precipitate crystals within 30 minutes to an hour;  and of course, one can culture as much of the algae as one wants, so even if viability is compromised, they would probably live long enough to start removing strontium!
> There could be concerns regarding passing up of Sr-90 along the food-chain [which could possibly be circumvented by restricting algal-growth within a specific area devoid of its natural predators].

Overall, this definitely appears to be a good idea to start with. Additionally, organismal-sources to sink-off other radioisotopes like plutonium, cobalt, cesium, iodine, etc. needs to be found out to come up with a robust radioactive-clean up regimen.

References

[1] Selective Sequestration of Strontium in Desmid Green Algae by Biogenic Co-precipitation with Barite. Minna R. Krejci, Lydia Finney, Stefan Vogt, and Derk Joester. DOI: 10.1002/cssc.201000448

[2] A. J. Brook, The Biology of Desmids, University of California Press, Berkeley,1981.

[3] M. Eisenbud, T. F. Gesell, Environmental Radioactivity from Natural, Industrial, and Military Sources, Academic Press, San Diego, 1997.

[4] M. Manos, N. Ding, M. Kanatzidis, Proc. Natl. Acad. Sci. USA 2008, 105, 3696; A. Braun et al., Application of Ion Exchange Processes for the Treatment of Radioactive Waste and Management of Spent Ion Exchangers, International Atomic Energy Agency, Vienna, 2002. 

[5] S. Singh, S. Eapen, V. Thorat, C. P. Kaushik, K. Raj, S. F. D’Souza, Ecotoxicol. Environ. Saf. 2008, 69, 306– 311.
[6] Nature news article

Circadian-Clocks tick without DNA

                            Intricate machinary of a clock. Is there a master-tuner? [image]

This idea would forever change the way we would look at Circadain-Rythms, simply because it trashed a dogma - one that required DNA to run biological-clocks!

Circadian Rythms: All forms of life undergo circadian (roughly 24-hour) fluctuations in energy availability that are tied to alternating cycles of light and darkness. These self-sustained rhythms could be biochemical, physiological, or behavioural  processes. Our biological clocks organize such internal energetic cycles through 'transcription–translation feedback loops'. 

What is known: There are specific "clock-genes" that comprise of a 'forward limb' involving a set of transcriptional activators that induce the transcription of a set of repressors which comprise the 'negative limb' and duly feeds back to inhibit the forward limb [1]. This modulatory cycle repeats itself every 24 hours. Energy-cycles show transcription-dependent circadian periodicity; such cycles include the alternating oxygenic and nitrogen-fixing phases of photosynthesis, and the glycolytic and oxidative cycles in eukaryotes. 

BIG Questions:

1.Is the nucleus [actually the DNA contained in it] necessary for clock-maintainance in mammals?

Actually, several classical model organisms that are genetically tractable (for example, yeast and C. elegans) have not been found to express any known ‘clock genes’, but do exhibit circadian rhythms [2,3].

2.Are the transcriptional [DNA-based] and nontranscriptional [non-DNA based] cycles interrelated?

Experiments:

** How do you check for the necessity [or not] of DNA in a given life process? Easy, check for the process in a cell type that lacks a nucleus.

Neill and Reddy did exactly that! They established human red blood cells (RBCs, enucleated-mammalian cells) as an appropriate model system to test if they might have a rythmic-clock operating. Crucially, these cells lack both a nucleus and the energy-producing mitochondria.

RBC's : non-nucleated and just perfect! [image]

These cells therefore function mainly as oxygen shuttles, utilizing haemoglobin. Interestingly, RBCs possess the evolutionarily conserved enzymes of the peroxiredoxin family [4], which control intarcellular peroxide leves and react to rising intracellular reactive oxygen species (ROS) levels by forming oligomers. Importantly, they had been previously showed to exhibit circadian-periodicity in hepatic-cells [5]

1] Do Peroxiredoxins exhibit circadian redox rhythms in RBC's?

O’Neill and Reddy monitored the monomer–dimer transition of peroxiredoxin proteins in RBCs from three human volunteers. The oligomerization-pattern was self-sustained over several cycles within an approximate 24-hour period.

Next, they had to prove that RBC's should show the property of 'Entrainment', i.e to be a useful timing mechanism, oscillations (of oscillators) should be tuneable by external cues so that they can be reset when misaligned. Here, they used Temperature as a cue. And indeed, Peroxiredoxin oxidation cycles were synchronized in response to temperature cycles.

* To rule out the presence of contaminating nucleated cells [WBC etc.], inhibitors of translation (Cycloheximide) and transcription (a-Amanitin) were added, which could not perturb the peroxiredoxin oxidation rhythm, proving that this clock could run efficiently in the absence of transcription and was totally independant of DNA.

As it turns out, in RBC's, Haemoglobin [Hb] is a major source of Peroxides via autooxidation [Heme structure, here on Wiki]. So, did Hb also show periodicity? Yes, it did! They used intrinsic front-face fluorescence as a real-time assay of rhythmicity [6] and indicated reversible low amplitude oxidation of haemoglobin in RBCs.

* Now, what drives the rhythmic cycles of oligomerization for peroxiredoxin? Are they related to othe Biochemical cycles on RBCs'?

Given that red blood cells are dependant on glycolysis for ATP synthesis, and this contributes significantly to the NADH flux in RBCs'. Could ATP and NADPH oscillate with cycle? Yes, Indeed! The researchers reported weak oscillations in the levels of ATP (and also NADPH)!

2] Could they be related to Nuclear events? Is there a connection between the nucleus's interior and exterior?

Experiments:

they assayed mouse embryonic fibroblasts [MEF] from Cry1/Cry2 double-knockout mice, which lack cyclical expression of known clock genes/proteins [7]. Rhythms in peroxiredoxin oxidation were altered relative to those seen in wild-type MEFs. Therefore, in nucleated cells, peroxiredoxin rhythms are influenced by the transcription–translation feedback loop.

* Could the reverse be true? That is, if levels of Peroredoxin in cells fall, could it effect levels of transcription?

Indeed, as knockdown of PRX2 and PRX4 in human U2OS-cells resulted in a long-period phenotype, whereas si-RNAs directed against PRX3 and PRX5 depressed the amplitude of circadian oscillations!
Therefore, in nucleated cells, there is likely to be an intricate interplay between transcription-dependent processes and non-nuclear events, which seem to be reciprocally regulating each other.

 Take Home message:

Circadian Rythms are NOT maintained  exclusively by the Nucleus, but are also influenced by events occuring in the cytosol, AND adequate modulation is brought about by an essential interplay interplay of both processes.

 

References

 

* This paper

O’Neill, J. S. & Reddy, A. B. Circadian clocks in human red blood cells. Nature doi:10.1038/nature09702

** the other paper [in the same issue of Nature by same group]
O’Neill, J. S. et al. Circadian rhythms persist without transcription in a eukaryote.Nature doi:10.1038/nature09654
 
1. Reddy, A. B. & O’Neill, J. S. Healthy clocks, healthy body, healthy mind. Trends Cell Biol. 20, 36–44 (2009).

2. Eelderink-Chen, Z. et al. A circadian clock in Saccharomyces cerevisiae. Proc. Natl Acad. Sci. USA 107, 2043–2047 (2010).

3. Kippert, F., Saunders, D. S. & Blaxter, M. L. Caenorhabditis elegans has a circadian clock. Curr. Biol. 12, R47–R49 (2002).

4. Hall, A., Karplus, P. A. & Poole, L. B. Typical 2-Cys peroxiredoxins–structures, mechanisms and functions. FEBS J. 276, 2469–2477 (2009).

5. Reddy, A. B. et al. Circadian orchestration of the hepatic proteome. Curr. Biol. 16, 1107–1115 (2006).

6. Kennett, E. C. et al. Investigation of methaemoglobin reduction by extracellular NADH in mammalian erythrocytes. Int. J.Biochem.Cell Biol.37,1438–1445(2005).

7. Yagita, K., Tamanini, F., van der Horst, G. T. J.&Okamura, H. Molecular mechanisms of the biological clock in cultured fibroblasts. Science 292, 278–281 (2001).

* c u r i o s i t a s *: Lazy Sperms Check Inbreeding

* c u r i o s i t a s *: Lazy Sperms Check Inbreeding

Lazy Sperms Check Inbreeding

Inbreeding (mating between relatives) is costly, primarily as a consequence of the expression of deleterious recessive alleles[1,2] from a overtly constricted gene-pool.

Some instances of adverse effects of inbreeding: The dwindling numbers of cheetahs has been attributed to a genetic bottleneck caused by heavy inbreeding [3], and in humans, appearance of several harmful traits (eg. Haemophilia in european royal families, 'reproductive wastage' in a population of Dammam, Saudi Arabia [4], Ellis-van Creveld  disease in Amish settlements [5], decreased fertility rates in Hutterites [6]  etc.) has been attributed to unabated consanguineous relationships.

Researchers have long suspected that polyandry - females taking multiple mates - evolved in some species as a strategy to reduce breeding with relatives, and therefore as a means to reduce the negative fitness consequences of mating with genetically related males.

Evidence suggests that the genetic relatedness (or genetic similarity) between mating partners is associated with competitive fertilization success [7]. In externally fertilizing fishes, it has been shown that the ovarian fluid (OF) released by the females with their eggs during spawning affects sperm swimming velocity [8], and this effect is influenced by the identity (genotype) of the interacting male and female [9]. Furthermore, there is ample evidence that sperm kinematic parameters are important determinants of sperm competition success [10].

Clelia Gasparini and Andrea Pilastro from the University of Padova, Italy, investigated female preference for unrelated mates in the guppy (Poecilia reticulata), an internally fertilizing species of fish, in which the females mate multiply.

Their aim was to experimentally demonstrate whether the selection bias which would determine mechanisms reducing fertilizations by genetically related mates was operating at the gametic (sperm and ova) level.

                                                  Guppies (source)

Reasons why Guppies were used as a model system:

1. ideal model to study post-copulatory mechanisms of inbreeding avoidance [11].

2. inbreeding has deleterious fitness consequences in this species [12].

3. males can undermine female choice by means of gonopodial thrusting [13], a coercive mating tactic that allows males to forcibly inseminate females.

4. Paternity analysis in natural populations has revealed a paternity skew towards unrelated males [14], suggesting that mechanisms reducing fertilizations by genetically related mates do operate in this species.

Experiments:

a. Paired sperm competition test in which sperms were artificially inseminated two unrelated females. The difference in paternity success across females in relation to the difference in genetic relatedness among mates was later analysed.

b. Determination of whether insemination from related males would result in a reduced brood size, via differential fertilization success or embryo viability, by comparing brood size of females that were artificially inseminated with the sperm from either a brother or an unrelated male.

c. Finally, exploration of physiological mechanisms by which females mediate inbreeding avoidance.

Critical Observations:

To confirm that polyandrous female guppies do give an edge to sperm from non-related males, Andrea Pilastro and Clelia Gasparini at the University of Padova in Italy performed artificial insemination of 28 virgin females with sperm from either an unrelated or related male.

* Related males fertilized 10 percent fewer eggs than unrelated males.

To then investigate the source of the advantage [expt. 'c'], the team looked at the interaction between sperm and OF.

In an in vitro computer assisted sperm analysis (commonly called CASA) measurement aimed at measuring critical kinematic parameters of sperm movement.
* A male guppy's sperm-velocity is 5-10 percent slower in the OF of his sister than that of an unrelated female.

In other words, it demonstrates that genetic relatedness influences the effect of OF on sperm performance by increasing the swimming velocity of sperm from unrelated males in the guppy. 

This throws up an important point, that there now exists an interesting coincidence between the effect measured in sperm velocity and that consequently found in paternity.

Future work / Possibilites:
How the guppies' genetics influence OF-sperm interaction is still unknown, but Pilastro suspects the involvement of signaling peptides/receptors etc. present on the sperm-surface.

Although it might be beyond the scope of this  investigation, but an 'In-vitro-fertilization' expt. (in the absense of 'OF') could have ruled out inherent inequalities in fertilization-capabilites of sperms from brothers and non-relative donors.

Although this may not be the only explanation for biased paternity, but it's an interesting paper since it has shown experimental evidence for this kind of selection for the first time. Indeed, it could also be a common mode of sexual selection among other species as well.

References


The paper: Clelia Gasparini and Andrea Pilastro, 2010. Cryptic female preference for genetically unrelated males is mediated by ovarian fluid in the guppy. Proceedings of the Royal Society. doi:10.1098/rspb.2010.2369


1. Thornhill, N.W. 1993 The natural history of inbreeding and outbreeding: theoretical and empirical perspectives. Chicago, IL: Chicago University Press.

2. Charlesworth, B. & Charlesworth, D. 1999 The genetic basis of inbreeding depression. Genet. Res. 74, 329–340

3. M Menotti-Raymond and S J O'Brien, 1993. Dating the genetic bottleneck of the African cheetah.  Proc Natl Acad Sci, 90(8): 3172–3176.

4. Al-Abdulkareem, A. and Ballal, S. 1998. Consanguineous Marriages in an Urban Area of Saudi Arabia: Rates and Adverse Health Effects on Offspring. Journal of Community Health, Vol. 23, No. 1, pp 75-83.

5. Dorsten, L., Hotchkiss, L., and King, T. 1999. The Effect of Inbreeding on Early Childhood Mortality: Twelve Generations of an Amish Settlement. Demography. Vol. 36. No. 2. pp. 263-271.

6. Ober, C., Hyslop, T., and Hauck, W. 1999. Inbreeding Effects on Fertility in Humans: Evidence For Reproductive Compensation. American Journal of Human Genetics. Vol. 64. pp 225-231.
7. Olsson, M., Shine, R., Madsen, T., Gullberg, A. & Tegelstrom, H. 1996 Sperm selection by females. Nature, 383, 585–585.

8. Urbach, D., Folstad, I. & Rudolfsen, G. 2005 Effects of ovarian fluid on sperm velocity in Arctic charr (Salvelinus alpinus). Behav. Ecol. Sociobiol. 57, 438–444.
9. Rosengrave, P., Gemmell,N. J.,Metcalf, V.,McBride, K.& Montgomerie, R. 2008 A mechanism for cryptic female choice in chinook salmon. Behav. Ecol. 19, 1179–1185.
10. Gage,M. J. G., Macfarlane, C. P., Yeates, S.,Ward, R. G., Searle, J. B. & Parker, G. A. 2004 Spermatozoal traits and sperm competition in Atlantic salmon: relative sperm velocity is the primary determinant of fertilization success. Current Biology. 14, 44–47.

11. Magurran, A. E. 2005 Evolutionary ecology: the Trinidadian guppy. Oxford, UK: Oxford University Press.

12. Zajitschek, S. R. K., Lindholm, A. K., Evans, J. P. & Brooks, R. C. 2009 Experimental evidence that high levels of inbreeding depress sperm competitiveness. J. Evolutionary Biology. 22, 1338–1345.

13. Pilastro, A. & Bisazza, A. 1999 Insemination efficiency of two alternative male mating tactics in the guppy (Poecilia reticulata). Proc. R. Soc. Lond. B 266, 1887–1891.

14. Johnson, A. M., Chappell, G., Price, A. C., Rodd, F. H., Olendorf, R. & Hughes, K. A. 2010 Inbreeding depression and inbreeding avoidance in a natural population of guppies (Poecilia reticulata). Ethology 116, 448–457.

* Slow sperm prevent inbreeding [story in 'The Scientist'] here.

* Insightful discussion on 'Inbreeding In Humans' [here].