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Larval therapy for leg ulcers (VenUS II): randomised controlled trial. BMJ 2009;338:b773
Objective: To compare the clinical effectiveness of larval therapy with a standard debridement technique (hydrogel) for sloughy or necrotic leg ulcers.
Design: Pragmatic, three armed randomised controlled trial.
Setting: Community nurse led services, hospital wards, and hospital outpatient leg ulcer clinics in urban and rural settings, United Kingdom.
Participants: 267 patients with at least one venous or mixed venous and arterial ulcer with at least 25% coverage of slough or necrotic tissue, and an ankle brachial pressure index of 0.6 or more.
Interventions: Loose larvae, bagged larvae, and hydrogel.
Main outcome measures: The primary outcome was time to healing of the largest eligible ulcer. Secondary outcomes were time to debridement, health related quality of life (SF-12), bacterial load, presence of meticillin resistant Staphylococcus aureus, adverse events, and ulcer related pain (visual analogue scale, from 0 mm for no pain to 150 mm for worst pain imaginable).
Results: Time to healing was not significantly different between the loose or bagged larvae group and the hydrogel group (hazard ratio for healing using larvae v hydrogel 1.13, 95% confidence interval 0.76 to 1.68; P=0.54). Larval therapy significantly reduced the time to debridement (2.31, 1.65 to 3.2; P<0.001). Health related quality of life and change in bacterial load over time were not significantly different between the groups. 6.7% of participants had MRSA at baseline. No difference was found between larval therapy and hydrogel in their ability to eradicate MRSA by the end of the debridement phase (75% (9/12) v 50% (3/6); P=0.34), although this comparison was underpowered. Mean ulcer related pain scores were higher in either larvae group compared with hydrogel (mean difference in pain score: loose larvae v hydrogel 46.74 (95% confidence interval 32.44 to 61.04), P<0.001; bagged larvae v hydrogel 38.58 (23.46 to 53.70), P<0.001).
Conclusions: Larval therapy did not improve the rate of healing of sloughy or necrotic leg ulcers or reduce bacterial load compared with hydrogel but did significantly reduce the time to debridement and increase ulcer pain.

Related:

• MRSA: Methicillin-resistant Staphylococcus aureus
• Novel treatments for MRSA
• Evolution and pathogenesis of Staphylococcus aureus

Lentiviruses are mammalian retroviruses known to infect cattle, cats, horses, sheep, and primates. They are the focus of intense study due to their causative association with AIDS in human. Although our knowledge on the origin and early evolution of HIV has grown exponentially over the past few years, much remains unresolved about the deeper relationships between primate and non-primate lentiviruses, the origin of lentiviruses, and their mode of structural evolution over long periods of evolutionary time. This is because these viruses evolve extremely rapidly, in a conflicting relationship with their hosts, and while their high mutation rate provides a wealth of information documenting their recent history, it also quickly erases evidence of their deeper ancestry. The lifecycle of retroviruses is atypical compared to other viruses in that after appropriate receptor recognition and entry in a specific cell type, their RNA genome is reverse transcribed into double-stranded DNA and integrated into the host genome as a provirus. Occasionally this process can take place in the host germline, and the integrated copy, also called endogenous retrovirus (ERV), may be transmitted vertically from parent to offspring and reach fixation in the host population. As such, ERVs constitute a fossil record of past viral infections that potentially provide an alternative way of gaining insights into the deep evolutionary history of present day exogenous retroviruses.

Although many ERVs have been characterized in mammals (e.g. 8% of the human genome), apparently very few derive from lentiviruses. Two reasons have traditionally been put forward to explain their absence in mammalian genomes: (i) they are of relatively recent evolutionary origin and endogenization has not yet commonly occurred, and/or (ii) they were not able to enter germ cells because of a very specific cell tropism. Recently however, an endogenous lentivirus called RELIK has been identified in the genome of rabbits and hares, whose germline integration was dated at least 12 millions years old. This discovery not only showed that lentiviruses were able to infiltrate mammalian germlines, but also demonstrated that this group of viruses is probably much older than what could previously be inferred based on sequence comparison of extant exogenous lentiviruses. New research now shows that a retrovirus related to HIV became stably integrated into the genomes of lemurs around 4.2 million years ago. The discovery of prosimian immunodeficiency virus (pSIV) offers new insights into the evolution of lentiviruses.

Based on “fossil” sequences collected from different lemur species, the researchers computationally reconstructed an apparently intact and complete DNA sequence for the ancestral prosimian lentivirus. The discovery that two different species of lemurs endemic to Madagascar suffered, independently and quasi-simultaneously, multiple germline infections of pSIV provides evidence that lentiviruses have repeatedly infiltrated the germline of prosimian species. These findings should allow future functional analysis of the extinct virus and advance our understanding of the biology of lentiviruses, including HIV. In addition, the characterization of this ancient lentivirus in lemurs raises the possibility that HIV-like retroviruses are still circulating today in the mammalian fauna of Madagascar.

Parallel Germline Infiltration of a Lentivirus in Two Malagasy Lemurs. 2009 PLoS Genet 5(3): e1000425
Retroviruses normally infect the somatic cells of their host and are transmitted horizontally, i.e., in an exogenous way. Occasionally, however, some retroviruses can also infect and integrate into the genome of germ cells, which may allow for their vertical inheritance and fixation in a given species; a process known as endogenization. Lentiviruses, a group of mammalian retroviruses that includes HIV, are known to infect primates, ruminants, horses, and cats. Unlike many other retroviruses, these viruses have not been demonstrably successful at germline infiltration. Here, we report on the discovery of endogenous lentiviral insertions in seven species of Malagasy lemurs from two different genera – Cheirogaleus and Microcebus. Combining molecular clock analyses and cross-species screening of orthologous insertions, we show that the presence of this endogenous lentivirus in six species of Microcebus is the result of one endogenization event that occurred about 4.2 million years ago. In addition, we demonstrate that this lentivirus independently infiltrated the germline of Cheirogaleus and that the two endogenization events occurred quasi-simultaneously. Using multiple proviral copies, we derive and characterize an apparently full length and intact consensus for this lentivirus. These results provide evidence that lentiviruses have repeatedly infiltrated the germline of prosimian species and that primates have been exposed to lentiviruses for a much longer time than what can be inferred based on sequence comparison of circulating lentiviruses. The study sets the stage for an unprecedented opportunity to reconstruct an ancestral primate lentivirus and thereby advance our knowledge of host–virus interactions.

Related:

• Phoenix from the ashes: The 5 million year old virus
• T Cell Responses to Human Endogenous Retroviruses in HIV-1 Infection
• Human endogenous retroviruses: from ancestral pathogens to bona fide genes
• HIV infection is due to the sins of the fathers
• Rooting the tree

PKR is a sentinel kinase constitutively expressed in all cells as an inactive protein that is subsequently activated by virus RNA produced during an infection. The active kinase perturbs virus replication by phosphorylating protein substrates in the cell. RNA helicase A (RHA) is a novel substrate for PKR. Viruses usurp this helicase to replicate their own genome. Phosphorylation of RHA by PKR perturbs the ability of the helicase to bind virus RNA. Correspondingly, PKR prevents the capacity of RHA to enhance expression of genetic elements encoded by the human immunodeficiency virus (HIV). In addition, HIV virions packaged within cells that also express protein fragments of RHA have enhanced infectivity. These fragments of RHA occur within a protein domain previously established to bind RNA but increasingly recognized to mediate protein–protein interactions. This supports an emerging role for these protein domains to coordinate the cell’s response to pathogen-associated RNA. The findings identify a new cell-signaling pathway important in the response to virus infection.

An Antiviral Response Directed by PKR Phosphorylation of the RNA Helicase A. 2009 PLoS Pathog 5(2): e1000311
The double-stranded RNA-activated protein kinase R (PKR) is a key regulator of the innate immune response. Activation of PKR during viral infection culminates in phosphorylation of the α subunit of the eukaryotic translation initiation factor 2 (eIF2α) to inhibit protein translation. A broad range of regulatory functions has also been attributed to PKR. However, as few additional PKR substrates have been identified, the mechanisms remain unclear. Here, PKR is shown to interact with an essential RNA helicase, RHA. Moreover, RHA is identified as a substrate for PKR, with phosphorylation perturbing the association of the helicase with double-stranded RNA (dsRNA). Through this mechanism, PKR can modulate transcription, as revealed by its ability to prevent the capacity of RHA to catalyze transactivating response (TAR)–mediated type 1 human immunodeficiency virus (HIV-1) gene regulation. Consequently, HIV-1 virions packaged in cells also expressing the decoy RHA peptides subsequently had enhanced infectivity. The data demonstrate interplay between key components of dsRNA metabolism, both connecting RHA to an important component of innate immunity and delineating an unanticipated role for PKR in RNA metabolism.

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• Viruses and Apoptosis

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• LabWork: Bacterial growth
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Many bacteria are facultative anaerobes, that is, they can grow in the presence or absence of O₂. In contrast to anaerobic respiration or fermentation, O₂ and aerobic respiration confer enormous energetic benefits on facultative bacteria by allowing the complete oxidation of a growth substrate and the concomitant conservation of much larger amounts of energy. Moreover, some energetically expensive processes such as nitrogen fixation are inhibited by O₂. Furthermore, hypoxic conditions are a signal to adopt a different “lifestyle” for some bacteria such as the dormant state of Mycobacterium tuberculosis associated with latent TB infections. The ability to adapt to changes in O₂ availability by expressing different groups of genes is controlled at the level of transcription by O₂-sensing regulatory proteins.

Bacterial sensors of oxygen. Curr Opin Microbiol. Feb 24 2009
The concentration of molecular oxygen (O₂) began to increase in the Earth’s atmosphere approximately two billion years ago. Its presence posed a threat to anaerobes but also offered opportunities for improved energy conservation via aerobic respiration. The ability to sense environmental O₂ thus became, and remains, important for many bacteria, both for protection and switching between anaerobic and aerobic respiration. Utilizing an iron–sulfur cluster as the sensor of O₂ exploits the ability of O₂ to oxidize the iron–sulfur cluster, ultimately resulting in cluster disassembly. When utilizing heme as the sensor, the capacity of O₂ to form a reversible Fe–O₂ bond or alternatively the oxidation of the heme iron atom itself is used to detect O₂ and switch regulators between active and inactive forms.

Related:

• Microbes and Oxygen
• The first appearance of eukaryotes and cyanobacteria

In the last week there has been some fairly wild speculation in the media about viruses which “cause” diabetes. The fuss came from the publication of a paper which claimed to have detected virus proteins in the pancreases of diabetes patients (The prevalence of enteroviral capsid protein vp1 immunostaining in pancreatic islets in human type 1 diabetes. Diabetologia 6 March 2009). At the same time, a separate study found four rare mutations in a gene which is thought to reduce the risk of developing type 1 diabetes and may be involved in the immune response to infection with enteroviruses (Rare Variants of IFIH1, a Gene Implicated in Antiviral Responses, Protect Against Type 1 Diabetes. Science Mar 5 2009).

The press was buzzing with speculation about the chances of a vaccine to prevent diabates. Very good news for diabetics? Well not so fast. Before we look at the science, let me tell you two things about myself. First, I have two close relatives who are affected by diabetes, so this is a disease I care a lot about. Second, I’ve been in the virology business a long time – and we’ve been here before.

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The new paper claimed to have detected “enterovirus capsid protein vp1″ in 44 out of 72 pancreases from children who had died of type-1 diabetes shortly after becoming ill, but in only three out of 50 neonatal and paediatric normal control specimens. Statistically there is a strong correlation in this study between diabetes and the presence of the virus protein, but a correlation does not indicate a cause. Are diabetics more susceptible to enterovirus infection? We don’t know. While it’s not ethically possible to satisfy Koch’s postulates in humans, we need to be very careful in inferring from small scale studies such as this one:

1. The microorganism must be found in abundance in all organisms suffering from the disease.
2. The microorganism must be isolated from a diseased organism and grown in pure culture.
3. The cultured microorganism should cause disease when introduced into a healthy organism.
4. The microorganism must be reisolated from the inoculated, diseased experimental host and identified as being identical to the original specific causative agent.

There are over a hundred different enteroviruses and the antibody used for detection of virus protein in this study (yes, that’s right, just one non-specific antibody) does not identify the virus involved. Vaccine against diabetes? I don’t think so.

But as I said, we’ve been here before. There are reports of viruses associated with diabetes dating from the 1960s, and a very well known model of Coxsackie virus B4 causing diabetes in mice dating from the 1970s (Coxsackie Viruses and Diabetes Mellitus. BMJ 1973 November 3; 4(5887): 260–262). So does the latest work add anything new, and is a vaccine against diabetes just around the corner? No. I wish it was.

Related:

• Can you have confidence in the news?
• Media coverage affects how people perceive the threat of disease

Related:

• Vaccination does not cause autism
• MMR: our children, our choice?

Alternative methods to antimicrobial chemotherapy for combating infectious disease do exist. In this article in Microbiology Today, Roy Sleator describes the role of probiotics in keeping pathogens at bay:

With life-cycles measured in minutes as opposed to years, bacteria have an extraordinary ability to evolve and adapt rapidly to changes in their environment. Thus, in a world where only the fittest survive, those bacteria which have developed resistance to antibiotics will predominate. This is particularly apparent in hospital environments where bacteria are in constant contact with many different antibiotics; such repeated exposure has facilitated the development of resistance to multiple antibiotics and what we now refer to as hospital acquired or nosocomial infections.

Read more

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• Probiotics – friendly bacteria?
• Probiotics – weapons in the war against gut pathogens
• Antibiotics, your gut, and you
• Gut Feeling

For over twenty years scientists have known that a normal protein in the brain, PrP, or prion protein, can become harmful and cause deadly illnesses like Creutzfeldt-Jakob disease (CJD) in humans, and bovine spongiform encephalopathy (BSE) in cattle. What they could not explain is why large amounts of this normal protein are produced by our bodies in the first place. In a new study, researchers reveal that PrP indeed plays a beneficial role for the organism – PrP helps cells communicate with one another during embryonic development.

In prion diseases, what transforms the normal PrP protein into a life-threatening substance is the abnormal alteration of its chemical structure. Moreover, prions have the treacherous ability to replicate by imprinting their abnormal structure into healthy PrPs, thereby generating new pathogenic particles. While this conversion process explains how prions are disseminated, an abnormal function of the prion protein is considered to be one of the reasons for neuronal degeneration. However, the normal function of PrP has remained an unsolved mystery for many years. All previous experiments in genetically modified mice had failed to provide conclusive evidence, as these animals lacking PrP seemed perfectly healthy. The scientists were able to show that the lack of PrP can cause clear physiological abnormalities in a living animal by using the tiny zebrafish as a model.

When the researchers microinjected zebrafish eggs with morpholinos, DNA-like molecules that prevent the normal production of PrP, the treated zebrafish embryos were unable to develop normally and eventually died. The proteins in the fish embryos normally found at cell-to-cell contact sites disappeared, rendering these cells unable to communicate and carry out the differentiation program that shapes the major structures of the body, including the nervous system. PrP serves as a glue element, bringing cells together and keeping them in contact. When two neighboring cells make contact, they become able to exchange important signals that affect the function of a tissue in the body. Although this work does not offer an immediate cure for CJD or BSE, it widens our understanding of prion diseases and provides hope for effective treatments.

Regulation of embryonic cell adhesion by the prion protein. 2009 PLoS Biol 7(3): e1000055
Prion proteins (PrPs) are key players in fatal neurodegenerative disorders, yet their physiological functions remain unclear, as PrP knockout mice develop rather normally. We report a strong PrP loss-of-function phenotype in zebrafish embryos, characterized by the loss of embryonic cell adhesion and arrested gastrulation. Zebrafish and mouse PrP mRNAs can partially rescue this knockdown phenotype, indicating conserved PrP functions. Using zebrafish, mouse, and Drosophila cells, we show that PrP: (1) mediates Caþ2-independent homophilic cell adhesion and signaling; and (2) modulates Caþ2-dependent cell adhesion by regulating the delivery of E-cadherin to the plasma membrane. In vivo time-lapse analyses reveal that the arrested gastrulation in PrP knockdown embryos is due to deficient morphogenetic cell movements, which rely on E-cadherin–based adhesion. Cell-transplantation experiments indicate that the regulation of embryonic cell adhesion by PrP is cell-autonomous. Moreover, we find that the local accumulation of PrP at cell contact sites is concomitant with the activation of Src-related kinases, the recruitment of reggie/flotillin microdomains, and the reorganization of the actin cytoskeleton, consistent with a role of PrP in the modulation of cell adhesion via signaling. Altogether, our data uncover evolutionarily conserved roles of PrP in cell communication, which ultimately impinge on the stability of adherens cell junctions during embryonic development.

Related:

• The Origin of BSE
• A novel approach in the molecular differentiation of prion strains
• What the heck are prions for?
• Alzheimer’s Disease and Prions