Transglutaminase type 2 plays a key role in the pathogenesis of Mycobacterium tuberculosis infection.

BACKGROUND: Mycobacterium tuberculosis (MTB), the aetiological agent of tuberculosis (TB), is capable of interfering with the phagosome maturation pathway, by inhibiting phagosome-lysosome fusion and the autophagic process to ensure survival and replication in macrophages. Thus, it has been proposed that the modulation of autophagy may represent a therapeutic approach to reduce MTB viability by enhancing its clearance. OBJECTIVE: The aim of this study was to investigate whether transglutaminase type 2 (TG2) is involved in the pathogenesis of MTB. RESULTS: We have shown that either genetic or pharmacological inhibition of TG2 leads to a marked reduction in MTB replicative capacity. Infection of TG2 knockout mice demonstrated that TG2 is required for MTB intracellular survival in macrophages and host tissues. The same inhibitory effect can be reproduced in vitro using Z-DON, a specific inhibitor of the transamidating activity of TG2. Massive cell death observed in macrophages that properly express TG2 is hampered by the absence of the enzyme and can be largely reduced by the treatment of wild-type macrophages with the TG2 inhibitor. Our data suggest that reduced MTB replication in cells lacking TG2 is due to the impairment of LC3/autophagy homeostasis. Finally, we have shown that treatment of MTB-infected murine and human primary macrophages with cystamine, a TG2 inhibitor already tested in clinical studies, causes a reduction in intracellular colony-forming units in human macrophages similar to that achieved by the anti-TB drug capreomycin. CONCLUSION: These results suggest that inhibition of TG2 activity is a potential novel approach for the treatment of TB.

Gene-expression analysis of cementoblasts and osteoblasts.

BACKGROUND AND OBJECTIVE: Cementum and bone are similar mineralized tissues, but cementum accumulates much more slowly than bone, does not have vasculature or innervation and does not undergo remodeling. Despite these differences, there are no well-established markers to distinguish cementoblasts from other mature mineralizing cells such as osteoblasts and odontoblasts. The purpose of this study was to assess differences in gene expression between cementoblasts and osteoblasts using gene profiling of cell populations isolated directly from osteocalcin-green fluorescent protein (OC-GFP) transgenic mice. MATERIAL AND METHODS: OC-GFP reporter mice were used as they show labeling of cementoblasts, osteoblasts and odontoblasts, but not of periodontal ligament fibroblasts, within the periodontium. We sorted cells digested from the molar root surface to isolate OC-GFP(+) cementoblasts. Osteoblasts were isolated from calvarial digests. Microarray analysis was performed, and selected results were confirmed by real-time PCR and immunostaining or in situ hybridization. RESULTS: Microarray analysis identified 95 genes that were expressed at least two-fold higher in cementoblasts than in osteoblasts. Our analysis indicated that the Wnt signaling pathway was differentially regulated, as were genes related to skeletal development. Real-time PCR confirmed that expression of the Wnt inhibitors Wnt inhibitory factor 1 (Wif1) and secreted frizzled-related protein 1 (Sfrp1) was elevated in cementoblasts compared with osteoblasts, and Wif1 expression was localized to the apical root region. In addition, the transcription factor BARX homeobox 1 (Barx1) was expressed at higher levels in cementoblasts, and immunohistochemistry indicated that BARX1 was expressed in apical cementoblasts and cementocytes, but not in osteoblasts or odontoblasts. CONCLUSION: The OC-GFP mouse provides a good model for selectively isolating cementoblasts, and allowed for identification of differentially expressed genes between cementoblasts and osteoblasts.

Bone-specific overexpression of NPY modulates osteogenesis.

OBJECTIVES: Neuropeptide Y (NPY) is a peptide involved in the regulation of appetite and energy homeostasis. Genetic data indicates that NPY decreases bone formation via central and peripheral activities. NPY is produced by various cell types including osteocytes and osteoblasts and there is evidence suggesting that peripheral NPY is important for regulation of bone formation. We sought to investigate the role of bone-derived NPY in bone metabolism. METHODS: We generated a mouse where NPY was over-expressed specifically in mature osteoblasts and osteocytes (Col2.3NPY) and characterized the bone phenotype of these mice in vivo and in vitro. RESULTS: Trabecular and cortical bone volume was reduced in 3-month-old animals, however bone formation rate and osteoclast activity were not significantly changed. Calvarial osteoblast cultures from Col2.3NPY mice also showed reduced mineralization and expression of osteogenic marker genes. CONCLUSIONS: Our data suggest that osteoblast/osteocyte-derived NPY is capable of altering osteogenesis in vivo and in vitro and may represent an important source of NPY for regulation of bone formation. However, it is possible that other peripheral sources of NPY such as the sympathetic nervous system and vasculature also contribute to peripheral regulation of bone turnover.

Temporal patterns of genetic and phenotypic variation in the epidemiologically important drone fly, Eristalis tenax.

Eristalis tenax L. (Diptera: Syrphidae) is commonly known as the drone fly (adult) or rat-tailed maggot (immature). Both adults and immature stages are identified as potential mechanical vectors of mycobacterial pathogens, and early-stage maggots cause accidental myiasis. We compared four samples from Mount Fruska Gora, Serbia, with the aim of obtaining insights into the temporal variations and sexual dimorphism in the species. This integrative approach was based on allozyme loci, morphometric wing parameters (shape and size) and abdominal colour patterns. Consistent sexual dimorphism was observed, indicating that male specimens had lighter abdomens and smaller and narrower wings than females. The distribution of genetic diversity at polymorphic loci indicated genetic divergence among collection dates. Landmark-based geometric morphometrics revealed, contrary to the lack of divergence in wing size, significant wing shape variation throughout the year. In addition, temporal changes in the frequencies of the abdominal patterns observed are likely to relate to the biology of the species and ecological factors in the locality. Hence, the present study expands our knowledge of the genetic diversity and phenotypic plasticity of E. tenax. The quantification of such variability represents a step towards the evaluation of the adaptive potential of this species of medical and epidemiological importance.

Costs and benefits of high mutation rates: adaptive evolution of bacteria in the mouse gut.

We have shown that bacterial mutation rates change during the experimental colonization of the mouse gut. A high mutation rate was initially beneficial because it allowed faster adaptation, but this benefit disappeared once adaptation was achieved. Mutator bacteria accumulated mutations that, although neutral in the mouse gut, are often deleterious in secondary environments. Consistently, the competitiveness of mutator bacteria is reduced during transmission to and re-colonization of similar hosts. The short-term advantages and long-term disadvantages of mutator bacteria could account for their frequency in nature.

Counteraction by MutT protein of transcriptional errors caused by oxidative damage.

Oxidized guanine (8-oxo-7,8-dihydroguanine; 8-oxo-G) is a potent mutagen because of its ambiguous pairing with cytosine and adenine. The Escherichia coli MutT protein specifically hydrolyzes both 8-oxo-deoxyguanosine triphosphate (8-oxo-dGTP) and 8-oxo-guanosine triphosphate (8-oxo-rGTP), which are otherwise incorporated in DNA and RNA opposite template A. In vivo, this cleaning of the nucleotide pools decreases both DNA replication and transcription errors. The effect of mutT mutation on transcription fidelity was shown to depend on oxidative metabolism. Such control of transcriptional fidelity by the ubiquitous MutT function has implications for evolution of RNA-based life, phenotypic expression, adaptive mutagenesis, and functional maintenance of nondividing cells.

Recombinant activated factor VII in refractory gastrointestinal haemorrhage of unknown aetiology.

We report a 27-year-old patient who suffered severe gastrointestinal (GI) bleeding of unknown aetiology after undergoing elective abdominal surgery to remove a colonic tumour. Although the immediate postoperative recovery period was uneventful, rectal bleeding and signs of haemorrhagic shock developed within 10 hours of surgery. Nasogastric aspiration and laparotomy failed to reveal the cause of the GI haemorrhage, and the patient remained unresponsive to conventional haemostatic therapy. Treatment with a single dose of recombinant activated factor VII (rFVIIa) 45 microg/kg led to reduced bleeding, improvements in haemodynamic status, and reduced transfusion requirements. Although further investigation is warranted, our findings suggest that rFVIla may be useful in the rescue treatment of severe GI haemorrhage of unknown origin (Tab. 1, Ref. 17).

Pediatric polytrauma at intensive care unit.

BACKGROUND: Polytrauma and its consequences present a rising diagnostic and therapeutic problem we face at ICU every day. The goal of this research was to analyze and improve the diagnostic and treatment procedures. PATIENTS AND METHODS: A prospective research carried out at the multidisciplinary Intensive Care Unit during a period of over 2 years included 126 patients aged less than 15 years. Immediately on admission, all patients received the necessary care strictly complying with polytrauma treatment algorithms. We recorded the patients basic data (age, sex), cause of injury (fall, traffic accidents etc.) and injury type (blunt or penetrating) as well as the immediately performed procedures. The analysis of patients and calculation of mortality rates was based on time that had elapsed from injury to arrival at ICU, and severity of their injuries assessed as ISS score. Injury severity was assessed on admission also by using GCS, PTS, NISS and TRISS. Performed surgical interventions, mechanical ventilation duration, and total ICU time were recorded, as well as the need for transfer to a pediatric trauma centre. RESULTS: 96 patients arrived within 2 hrs from injury, with ISS>15, and/or PTS<8. 52 patients arrived within "golden hour"; 37 of them had ISS 16-26 and 15 had ISS>26 with mortality rate 3 (8%) vs 3 (20%) (p<0.001). Of the remaining 44 patients, 30 had ISS 16-26 and 14 had ISS>26, with mortality rate 7 (23.3%) vs. 5 (35.7%) (p<0.001). CONCLUSION: The arrival at ICU during "golden hour", precise algorithms, high quality of diagnosis, monitoring and therapeutic procedures had an essential influence on the positive end-outcome and improved the survival and recovery rates in polytraumatized children (Tab. 4, Fig. 1, Ref. 18). Full Text (Free, PDF) www.bmj.sk.

Effects of APACHE II score on mechanical ventilation; prediction and outcome.

AIM: To evaluate the influence of Acute Physiology and Chronic Health Evaluation (APACHE II) score on the choice of mechanical ventilation method and treatment outcome. METHODS: A prospective, randomized trial was carried out at the multidisciplinary Intensive Care Unit over 22 months. Research sample consisted of 129 patients who required mechanical ventilation, divided in two groups: APACHE II < or = 20 and APACHE II > 20. Both groups were than randomized for either noninvasive or invasive mechanical ventilation. Comparison was made based on patient characteristics, objective parameters and influence of APACHE II score on treatment success and failure. RESULTS: APACHE II scoring was shown to have statistical significance on outcome assessment. Statistical significance was in favour of patients with APACHE II score < or = 20 vs > 20 (ventilator associated pneumonia 0 vs. 10, tracheotomy 0 vs. 16, Intensive Care Unit mortality 0 vs 12). Furthermore, in the group with APACHE II score > 20, after randomization, there was a statistical significance in favour of noninvasive mechanical ventilation in need for tracheotomy 2 (4%) vs. 14 (28%) (p < 0.001). CONCLUSION: Using good patient selection and applying strict protocols, in the group of patients with APACHE II < or = 20 all patients had successful mechanical ventilation, while in the group of patients with APACHE II > 20, noninvasive mechanical ventilation can be applied.

Molecular Biology Digest of Cell Mitophagy.

The homeostasis of eukaryotic cells relies on efficient mitochondrial function. The control of mitochondrial quality is framed by the combination of distinct but interdependent mechanisms spanning biogenesis, regulation of dynamic network, and finely tuned degradation either through ubiquitin-proteasome system or autophagy (mitophagy). There is continuous evolution on the pathways orchestrating the mitochondrial response to stress signals and the organelle adaptation to quality control during acute and subtle dysfunctions. Notably, it remains indeed ill-defined whether active mitophagy leads to cell survival or death by defective mitochondrial degradation. Above all, uncharted is whether and how pharmacologically tackle these mechanisms may lead to conceive novel therapeutic strategies for treating conditions associated with the defective mitochondria. Here, we attempt to provide a chronological and comprehensive overview of the determining discoveries, which have led to the current knowledge of mitophagy.

The rise and fall of mutator bacteria.

Bacteria with elevated mutation rates are frequently found among natural isolates. This is probably because of their ability to generate genetic variability, the substrate for natural selection. However, such high mutation rates can lead to the loss of vital functions. The evolution of bacterial populations may happen through alternating periods of high and low mutation rates. The cost and benefits of high mutation rates in the course of bacterial adaptive evolution are reviewed.

Genetic barriers among bacteria.

Barriers to chromosomal gene transfer between bacterial species control their genetic isolation. These barriers, such as different microhabitats, the host ranges of genetic exchange vectors and restriction-modification systems, limit gene exchange, but the major limitation is genomic sequence divergence. The mismatch-repair system inhibits interspecies recombination, the inducible SOS system stimulates interspecies recombination, while natural selection determines the effective recombination frequencies.

Structure of recombinants from conjugational crosses between Escherichia coli donor and mismatch-repair deficient Salmonella typhimurium recipients.

To get more insight into the control of homologous recombination between diverged DNA by the Mut proteins of the long-patch mismatch repair system, we have studied interspecies Escherichia coli/Salmonella typhimurium recombination. Knowing that the same recombination pathway (RecABCD) is responsible for intraspecies and interspecies recombination, we have now studied the structure (replacement vs. addition-type or other rearrangement-type recombinants) of 81 interspecies recombinants obtained in conjugational crosses between E. coli donor and mutL, mutS, mutH, mutU or mut+ S. typhimurium recipients. Taking advantage of high interspecies sequence divergence, a physical analysis was performed on one third of the E. coli Hfr genome, which was expected to be transferred to S. typhimurium F- recipients during 40 min before interruption of the mating. Probes specific for each species were hybridized on dot blots of genomic DNA, or on colonies, and the composition of the rrn operons was determined from purified genomic DNA. With very few exceptions, the structure of these interspecies recombinants corresponds to replacements of one continuous block of the recipient genome by the corresponding region of the donor genome.

Genetic variability and adaptation to stress.

Besides an immediate cellular adaptation to stress, organisms can resist such challenges through changes in their genetic material. These changes can be due to mutation or acquisition of pre-evolved functions via horizontal transfer. In this chapter we will review evidence from bacterial genetics that suggests that the frequency of such events can increase in response to stress by activating mutagenic response (e.g. the SOS response) and by inhibiting antimutagenic activities (e.g. mismatch repair system, MRS). Natural selection, by favoring adaptations, can also select for the mechanism(s) that has/have generated the adaptive changes by hitchhiking. These mutator mechanisms can sometimes respond very specifically, though blindly, to the challenge of the environment. Such stress-induced increases in mutation rates enhance genetic polymorphism, which is the structural component of the barrier to genetic exchange. Since SOS and MRS are the enzymatic controls of this barrier, the modulation of these systems can lead to a burst of speciation.

Evolution-driving genes.

Genomic sequences provide evidence for a common origin of life and its evolution via selection of genetic variants created by mutation and recombination. Two classes of genes are known to accelerate mutation and/or recombination rates in bacterial populations: stress-inducible wild-type genes, usually part of the SOS regulon, and genes whose functional loss, or downregulation, increases the rate of genetic variability (mutator and/or hyper-rec mutants).

Second-order selection in bacterial evolution: selection acting on mutation and recombination rates in the course of adaptation.

The increase in genetic variability of a population can be selected during adaptation, as demonstrated by the selection of mutator alleles. The dynamics of this phenomenon, named second-order selection, can result in an improved adaptability of bacteria through regulation of all facets of mutation and recombination processes.

Evolution of evolvability.

Genomic sequence data provide evidence for a common origin of life and for its evolution by genetic variation via mutation and recombination. This paper discusses the fundamental dialectic paradigm of evolution--stability versus variability--at the crossroads of molecular genetics, population genetics, ecology, and the emerging science of experimental evolution. Experimental evolution of molecules, viruses, and bacteria can be used not only to test some basic evolutionary hypotheses but also to create new organisms for applications in biotechnology, agriculture, and medicine.

Repair of cytotoxic lesions induced by N-methyl-N'-nitro-N-nitrosoguanidine in Salmonella typhimurium and Escherichia coli.

The role of nucleotide excision repair and 3-methyladenine DNA glycosylases in removing cytotoxic lesions induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in Salmonella typhimurium and Escherichia coli cells was examined. Compared to the E. coli wild-type strain, the S. typhimurium wild-type strain was more sensitive to the same dose of MNNG. Nucleotide excision repair in both bacterial species does not contribute significantly to the survival after MNNG treatment, indicating that the observed differences in survival between S. typhimurium and E. coli should be attributed to DNA-repair systems other than nucleotide excision repair. The survival of the E. coli alkA mutant strain is seriously affected by the lack of 3-methyladenine DNA glycosylase II, accentuating the importance of this DNA-repair enzyme in protecting E. coli cells against the lethal effects of methylating agents. Following indications from our experiments, the existence of an alkA gene analogue in S. typhimurium has been questioned. Dot-blot hybridisation, using the E. coli alkA gene as a probe, was performed, and such a nucleotide sequence was not detected on S. typhimurium genomic DNA. The existence of constitutive 3-methyladenine DNA glycosylase, analogous to the E. coli Tag gene product in S. typhimurium cells, suggested by the results is discussed.

The influence of the nucleotide excision-repair system on mutagenesis in Salmonella typhimurium LT2 after exposure to low doses of monofunctional alkylating agents.

The role of nucleotide excision repair in the mutagenicity of the monofunctional alkylating agents N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), methyl methanesulfonate (MMS), N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG), and N-ethyl-N-nitrosourea (ENU) in Salmonella typhimurium was examined. The mutagenic potential of the mutagenic agents used increased in the following order: MMS less than ENU less than ENNG less than MNNG. The results obtained confirm the involvement of nucleotide excision repair in the removal of mutagenic lesions from the DNA of S. typhimurium cells exposed to high doses of methylating as well as ethylating agents. At the low doses of all the alkylating agents used, the nucleotide excision repair-proficient strain was mutagenized more efficiently than the uvrB mutant. This phenomenon, a consequence of competition between nucleotide excision-repair enzymes and constitutive O6-methylguanine-DNA methyltransferase, is discussed.