A dynamic and intricate regulatory network determines Pseudomonas aeruginosa virulence

Pseudomonas aeruginosa is a metabolically versatile bacterium that is found in a wide range of biotic and abiotic habitats. It is a major human opportunistic pathogen causing numerous acute and chronic infections. The critical traits contributing to the pathogenic potential of P. aeruginosa are the production of a myriad of virulence factors, formation of biofilms and antibiotic resistance. Expression of these traits is under stringent regulation, and it responds to largely unidentified environmental signals. This review is focused on providing a global picture of virulence gene regulation in P. aeruginosa. In addition to key regulatory pathways that control the transition from acute to chronic infection phenotypes, some regulators have been identified that modulate multiple virulence mechanisms. Despite of a propensity for chaotic behaviour, no chaotic motifs were readily observed in the P. aeruginosa virulence regulatory network. Having a ‘birds-eye’ view of the regulatory cascades provides the forum opportunities to pose questions, formulate hypotheses and evaluate theories in elucidating P. aeruginosa pathogenesis. Understanding the mechanisms involved in making P. aeruginosa a successful pathogen is essential in helping devise control strategies.     

Development of Standardized Insulin Treatment Protocols for Spontaneous Rodent Models of Type 1 Diabetes

Standardized protocols for maintaining near-normal glycemic levels in diabetic rodent models for testing therapeutic agents to treat disease are unavailable. We developed protocols for 2 common models of spontaneous type 1 diabetes, the BioBreeding diabetes-prone (BBDP) rat and nonobese diabetic (NOD) mouse. Insulin formulation, dose level, timing of dose administration, and delivery method were examined and adjusted so that glycemic levels remained within a normal range and fluctuation throughout feeding and resting cycles was minimized. Protamine zinc formulations provided the longest activity, regardless of the source of insulin. Glycemic control with few fluctuations was achieved in diabetic BBDP rats through twice-daily administration of protamine zinc insulin, and results were similar regardless of whether BBDP rats were acutely or chronically diabetic at initiation of treatment. In contrast, glycemic control could not be attained in NOD mice through administration of insulin twice daily. However, glycemic control was achieved in mice through daily administration of 0.25 U insulin through osmotic pumps. Whereas twice-daily injections of protamine zinc insulin provided glycemic control with only minor fluctuations in BBDP rats, mice required continuous delivery of insulin to prevent wide glycemic excursions. Use of these standard protocols likely will aid in the testing of agents to prevent or reverse diabetes.Abbreviations: BBDP, BioBreeding diabetes-prone; BBDR, BioBreeding diabetes-resistant; NOD, nonobese diabetic; PZI, protamine zinc insulin; T1D, type 1 diabetes; VAF, viral-antibody–free; ZT, Zeitgeber timeClinical trials to prevent or reverse type 1 diabetes (T1D) are predicated on preclinical study data obtained from animal models of the disease to determine agents that exhibit efficacy and translational potential. However, according to findings published over the past several years (summarized in references 2, 17, and 31), not all preclinical T1D studies are created equal. Without a standardized screening process, the hundreds of candidate therapeutic agents in development cannot be evaluated critically for translational potential. One parameter that varies considerably from report to report in T1D reversal studies is the insulin treatment provided to diabetic NOD mice. To address the need for standardized preclinical screening of new therapeutics, the National Institute for Diabetes and Digestive and Kidney Diseases has developed the Type 1 Diabetes Preclinical Testing Program.^2,35 Under this program, a central contract testing facility (Biomedical Research Models) bridged the gap between discovery of potential therapeutics and clinical testing for efficacy in prevention or reversal of T1D. Using 2 of the best characterized models of T1D, the BioBreeding diabetes-prone (BBDP) rat and the nonobese diabetic (NOD) mouse, we sought to develop standardized protocols for the treatment of diabetes with insulin to provide the best glycemic control throughout the fed and nonfed states. We began by housing these models in a viral-antibody–free (VAF) barrier facility, we created study designs approved by a scientific advisory board consisting of leaders in the field, and we performed studies by using standard operation procedures.The standard of care in patients with T1D is to attempt to maintain near-normal glucose levels, by providing exogenous insulin therapy several times daily via injection or pump after rigorous monitoring of glycemic levels and by appropriately coordinating insulin dosing with food intake. Current blood glucose control in diabetic rodent models focuses on maintaining the diabetic animal in a state of moderate hyperglycemia, with normal weight gain in the absence of severe ketonuria. This state is achieved by once-daily injections of titrated insulin doses or by implantation of continuous release insulin pellets;³⁸ however, insulin types and methods can vary widely between institutions and laboratories, yielding a wide range of glycemic control. Therefore there is marked difference between the stringent glycemic control targeted by humans with diabetes as compared with the relatively loose glycemic control afforded to rodents with diabetes. Despite the many physiologic differences between humans and rodents, glycemic control potentially can be addressed by making insulin treatment in rodents more comparable in terms of glycemic control to what is achieved currently in humans, especially given that patients with T1D will continue to administer insulin during treatment with therapeutic agents (for example, antiCD3).¹¹ The lessening of the frequency, duration, and severity of hyperglycemic events is anticipated to provide the best chance for β cells to rest (function properly) while interventions are tested.²¹ Ideally, for these studies, animals should receive sufficient insulin to maintain glycemic levels close to the normal range in control nondiabetic animals.For these studies, we focused on the 2 most widely used spontaneous rodent models of T1D: the BioBreeding diabetes-prone (BBDP) rat and the nonobese diabetic (NOD) mouse.^1,12 The BBDP strain originated from a colony of outbred Wistar rats that developed spontaneous diabetes at the BioBreeding Laboratories in the 1970s. In the 1980s, the strain was acquired by the University of Massachusetts Medical School. During inbreeding, the BioBreeding diabetes- resistant (BBDR) control strain was established. Both strains are maintained at our facility and represent the most fully inbred (more than 110 generations) and characterized colonies available. BBDP rats develop T1D at 50 to 90 d of age at a frequency of approximately 85% to 90%, with equal frequency in male and female rats; the disease in BBDP rats results from autoimmune insulitis that is mediated primarily by CD4⁺ and CD8⁺ T cells and the development of autoantibodies to islet antigen. This insulitis is similar to that in human patients.¹⁸ Insulin therapy is required shortly after onset of hyperglycemia or death will occur due to ketoacidosis.¹⁹ The Gimap5 mutation in BBDP rats results in a T-cell lymphopenia and is necessary for development of T1D in BBDP rats (along with expression of a MHC class II RT1 B/D^u allele); adoptive transfer of splenocytes or regulatory T cells from BBDR rats before 35 d of age prevents the onset of diabetes in BBDP rats.^9,28,38 Alternatively, depletion of regulatory T cells from BBDR rats (which are nonlymphopenic) induces T1D in that strain.The NOD mouse strain originated from selective inbreeding of the Cataract Shionogi mouse strain and was imported from Japan to The Joslin Diabetes Center in 1984. NOD mice are now the most widely used preclinical model of T1D, in part due to the availability of genetic analysis and manipulation as well as the wide array of reagents available for mechanistic studies. The most commonly cited source for NOD mice is The Jackson Laboratory (Bar Harbor, ME), where female NOD mice develop disease at a frequency of 65% to 100% by 30 wk of age, whereas male NOD mice develop disease at a frequency of 35% to 85% (inbred for more than 83 generations). The incidence can vary from year to year³⁴ and from facility to facility depending on several factors, the most important being housing conditions.^15,26 The incidence of T1D in female NOD mice at our VAF barrier facility has been 65% to 80% over the past 3 y; this frequency can be far lower in nonVAF facilities. Diabetic NOD mice exhibit mild ketoacidosis, which allows them to survive for as long as several weeks after the onset of hyperglycemia without supportive insulin treatment. NOD mice also present with insulin resistance and a distinct stage of insulitis, referred to as peri-insulitis, that is not found in either human T1D or in diabetic BBDP rats.^5,18 Although both NOD mouse and BBDP rat models of T1D have particular advantages and disadvantages, a prudent path of drug development would include the examination of the therapeutic efficacy of novel agents in both models.^2,31To standardize and improve current testing protocols, we developed insulin treatment regimens that maintain blood glucose levels near normal levels throughout day and night activities over prolonged periods, as would be expected to occur in interventional clinical trials. We show here that whereas 2 daily injections of insulin to diabetic BBDP rats were sufficient to achieve our goal, diabetic NOD mice required continuous delivery of insulin through the implantation of osmotic pumps.     

Genetic Variation in Healthy Oldest-Old

Individuals who live to 85 and beyond without developing major age-related diseases may achieve this, in part, by lacking disease susceptibility factors, or by possessing resistance factors that enhance their ability to avoid disease and prolong lifespan. Healthy aging is a complex phenotype likely to be affected by both genetic and environmental factors. We sequenced 24 candidate healthy aging genes in DNA samples from 47 healthy individuals aged eighty-five years or older (the ‘oldest-old’), to characterize genetic variation that is present in this exceptional group. These healthy seniors were never diagnosed with cancer, cardiovascular disease, pulmonary disease, diabetes, or Alzheimer disease. We re-sequenced all exons, intron-exon boundaries and selected conserved non-coding sequences of candidate genes involved in aging-related processes, including dietary restriction (PPARG, PPARGC1A, SIRT1, SIRT3, UCP2, UCP3), metabolism (IGF1R, APOB, SCD), autophagy (BECN1, FRAP1), stem cell activation (NOTCH1, DLL1), tumor suppression (TP53, CDKN2A, ING1), DNA methylation (TRDMT1, DNMT3A, DNMT3B) Progeria syndromes (LMNA, ZMPSTE24, KL) and stress response (CRYAB, HSPB2). We detected 935 variants, including 848 single nucleotide polymorphisms (SNPs) and 87 insertion or deletions; 41% (385) were not recorded in dbSNP. This study is the first to present a comprehensive analysis of genetic variation in aging-related candidate genes in healthy oldest-old. These variants and especially our novel polymorphisms are valuable resources to test for genetic association in models of disease susceptibility or resistance. In addition, we propose an innovative tagSNP selection strategy that combines variants identified through gene re-sequencing- and HapMap-derived SNPs.     

Two amphibian diseases, chytridiomycosis and ranaviral disease, are now globally notifiable to the World Organization for Animal Health (OIE): an assessment

The global trade in amphibians entails the transport of tens of millions of live animals each year. In addition to the impact harvesting wild animals can have on amphibian populations, there is mounting evidence that the emerging pathogens Batrachochytrium dendrobatidis and ranaviruses, the aetiological agents of chytridiomycosis and ranaviral disease, respectively, are spread through this trade. The link between these pathogens and amphibian declines and extinctions suggests that the epidemiological impact of the trade is significant and may negatively affect conservation and trade economics. Here we present a brief assessment of the volume of the global trade in live amphibians, the risk of individuals harboring infection, and information on the recent listing by the World Organization for Animal Health (OIE) of chytridiomycosis and ranaviral disease in the OIE Aquatic Animal Health Code. This listing made chytridiomycosis and ranaviral disease internationally notifiable diseases and thus subject to OIE standards, which aim to assure the sanitary safety of international trade in live amphibians and their products.     

Inhibitors of HIV-1 attachment. Part 4: A study of the effect of piperazine substitution patterns on antiviral potency in the context of indole-based derivatives

4-Fluoro- and 4-methoxy-1-(4-benzoylpiperazin-1-yl)-2-(1H-indol-3-yl)ethane-1,2-dione (2 and 3, respectively) have been characterized as potent inhibitors of HIV-1 attachment that interfere with the interaction of viral gp120 with the host cell receptor CD4. As part of an effort to understand fundamental aspects of this pharmacophore, discovered originally using a high throughput cell-based screen, modification and substitution of the piperazine ring was examined in the context of compounds 6a–ah. The piperazine ring was shown to be a critical element of the HIV-1 attachment inhibiting pharmacophore, acting as a scaffold to deploy the indole glyoxamide and benzamide in a topographical relationship that complements the binding site on gp120.     

The secretion antigen SA5K has a role in the adaptation of Mycobacterium bovis bacillus Calmette-Guérin to intracellular stress and hypoxia

The Mycobacterium tuberculosis TB8.4 (Rv1174c) gene encodes a secreted protein of 8.4 kDa (TB8.4) which has been suggested to be involved in reactivation of dormant mycobacteria. We have previously reported that inactivation of an identical gene (sa5k) in Mycobacterium bovis BCG causes impaired ability of the mutant strain (BCGsa5k::aph) to grow inside human macrophages. This study aimed to investigate the role of TB8.4 in the reactivation of aged cultures of BCG as well as the role of the sa5k gene in the resistance of BCG to intracellular stress conditions and adaptation to hypoxia. Although when added to aged cultures of BCG, TB8.4 caused a statistically significant increase in the number of colony-forming units, a similar effect was obtained in cultures incubated with BSA, suggesting a non-specific growth stimulation by TB8.4. Compared to parental BCG, the BCGsa5k::aph strain showed an increased susceptibility to reactive oxygen and nitrogen intermediates and to acid stress and an impaired ability to adapt to reduced O2 concentrations, when tested in the oxygen-limited Wayne culture system. These results suggest that the product of the sa5k gene (SA5K protein) has a role in both resistance of BCG to intracellular stress and in its adaptation to hypoxia.     

The Decline of the Sharp-Snouted Day Frog (Taudactylus acutirostris): The First Documented Case of Extinction by Infection in a Free-Ranging Wildlife Species?

Infectious diseases are increasingly recognized as the cause of mass mortality events, population declines, and the local extirpation of wildlife species. In a number of cases, it has been hypothesized that pathogens have caused species extinctions in wildlife. However, there is only one definitively proven case of extinction by infection, and this was in a remnant captive population of a Polynesian tree snail. In this article, we review the potential involvement of infectious disease in the recent extinction of the sharp-snouted day frog Taudactylus acutirostris. Our review of available evidence suggests that a virulent pathogen of amphibians, Batrachochytrium dendrobatidis, caused a rapid, catastrophic decline of this species, from which it did not recover. We propose that this is the first case of extinction by infection of a free-ranging wildlife species where disease acted as both the proximate and ultimate cause of extinction. This highlights a probable underreporting of infectious disease as a cause of biodiversity loss historically and currently.     

Generation and maintenance of diversity in the cattle MHC class I region

Major histocompatibility complex (MHC) class I genes play a crucial role in the immune defence against intracellular pathogens. An important evolutionary strategy is to generate and maintain a high level of diversity in these genes. Humans express three highly polymorphic classical MHC class I genes (HLA-A, HLA-B and HLA-C). In contrast, some species, for example rat and rhesus macaque, maintain diversity by generation of haplotypes that vary considerably with regard to the number and combination of transcribed genes. Cattle appear to use both strategies. We show that various combinations of six apparently classical genes, three of which are highly polymorphic, are transcribed on different haplotypes. Although additional sequences were identified in both cDNA and gDNA, it was not possible to assign them to any of these defined genes. Most were highly divergent or were non-classical class I genes. Thus, we found little evidence for frequent duplication and deletion of classical class I genes as reported in some other species. However, the maintenance of class I diversity in cattle may involve limited gene shuffling and deletion, possibly as a result of unequal crossing-over within the class I region.The first two authors made an equal contribution to this work.