The soil fungus Trichoderma harzianum has been shown to act as a mycoparasite against a range of economically important aerial and soil-borne plant pathogens, being successfully used in the field and greenhouse (1-3). Different factors involved in the antagonistic properties of Trichoderma have been identified, including antibiotics (4-8) and hydrolytic enzymes such as 1,3-3-glucanases, proteases, and chitinases (9).The interaction between Trichoderma and its host is first detectable as chemotropic growth of hyphae of the mycoparasite toward its host (10,11). When the mycoparasite reaches the host, its hyphae often coil around it or are attached to it by forming hook-like structures (12)(13)(14)(15)(16). After these interactions, the mycoparasite penetrates the host mycelium, apparently by partially degrading its cell wall (12,13). Microscopic observations (6,10,13,(17)(18)(19) suggest that Trichoderma spp. produces and excretes mycolytic enzymes, as indicated by localized cell wall lysis at points of interaction. The susceptible host hyphae show rapid vacuolation, collapse, and disintegration (10). Thus, the mycoparasitic process involves (i) chemotropic growth of Trichoderma, (ii) recognition of the host by the mycoparasite and attachment, (iii) excretion of extracellular enzymes, (iv) hyphae penetration, and (v) lysis of the host.Chitin and 1,3-3-glucan are the two major structural components of the cell wall of many plant pathogenic fungi.Therefore, it is expected that the 1,3-3-glucanases, chitinases, and proteases produced extracellularly by Trichoderma (18,(20)(21)(22)(23) play an important role in biocontrol.

The endochitinase gene chiA74 from Bacillus thuringiensis serovar kenyae strain LBIT-82 was cloned in Escherichia coli DH5␣F. A sequence of 676 amino acids was deduced when the gene was completely sequenced. A molecular mass of 74 kDa was estimated for the preprotein, which includes a putative 4-kDa signal sequence located at the N terminus. The deduced amino acid sequence showed high degree of identity with other chitinases such as ChiB from Bacillus cereus (98%) and ChiA71 from Bacillus thuringiensis serovar pakistani (70%). Additionally, ChiA74 showed a modular structure comprised of three domains: a catalytic domain, a fibronectin-like domain, and a chitin-binding domain. All three domains showed conserved sequences when compared to other bacterial chitinase sequences. A ca. 70-kDa mature protein expressed by the cloned gene was detected in zymograms, comigrating with a chitinase produced by the LBIT-82 wild-type strain. ChiA74 is active within a wide pH range (4 to 9), although a bimodal activity was shown at pH 4.79 and 6.34. The optimal temperature was estimated at 57.2°C when tested at pH 6. The potential use of ChiA74 as a synergistic agent, along with the B. thuringiensis insecticidal Cry proteins, is discussed.Bacillus thuringiensis is an insecticidal bacterium whose activity is based on the effect of single or mixed Cry or Cyt proteins, acting additively or synergistically, although antagonism has also been reported (9). Despite reports of more than 3,000 insect species, within 16 orders and susceptible to different B. thuringiensis toxins, important pests are highly susceptible to only a few toxins (14). Additionally, development of resistance to Cry proteins, their slow mode of action, and the requirement of ingestion, along with some other limitations, justify the search for new approaches to improve the conventional use of B. thuringiensis.The insect midgut is internally coated by the peritrophic membrane, whose structure is basically composed of proteins and reinforced with chitin fibers. This is a physical barrier to bacterial or viral infections but allows the flow of digested nutrients, minerals, and water towards the midgut epithelium. It is known that the approach of ␦-endotoxins of B. thuringiensis to the microvillus receptor is highly facilitated when the peritrophic membrane is damaged or degraded, causing an increase in its insecticidal activity (25). Factors that damage the peritrophic membrane, such as the enhancin of granuloviruses, promote the insecticidal activity of commercial formulations of B. thuringiensis, especially on those pests with relatively low natural susceptibility, such as Helicoverpa zea (Boddie) and Spodoptera exigua (Hübner) (16). Likewise, the same effect is observed when Cry proteins are tested along with wild-type (31) or recombinant (11) chitinolytic bacteria, as well as with unpurified chitinase (Chi) (25,40).On the other hand, chitinases from B. thuringiensis are poorly studied (2, 7, 15), although interest has slowly increased due to their potential role as...

The Cuatro Cié negas Basin (CCB) in the central part of the Chihuahan desert (Coahuila, Mexico) hosts a wide diversity of microorganisms contained within springs thought to be geomorphological relics of an ancient sea. A major question remaining to be answered is whether bacteria from CCB are ancient marine bacteria that adapted to an oligotrophic system poor in NaCl, rich in sulfates, and with extremely low phosphorus levels (<0.3 M). Here, we report the complete genome sequence of Bacillus coahuilensis, a sporulating bacterium isolated from the water column of a desiccation lagoon in CCB. At 3.35 Megabases this is the smallest genome sequenced to date of a Bacillus species and provides insights into the origin, evolution, and adaptation of B. coahuilensis to the CCB environment. We propose that the size and complexity of the B. coahuilensis genome reflects the adaptation of an ancient marine bacterium to a novel environment, providing support to a ''marine isolation origin hypothesis'' that is consistent with the geology of CCB. This genomic adaptation includes the acquisition through horizontal gene transfer of genes involved in phosphorous utilization efficiency and adaptation to high-light environments. The B. coahuilensis genome sequence also revealed important ecological features of the bacterial community in CCB and offers opportunities for a unique glimpse of a microbe-dominated world last seen in the Precambrian.evolution ͉ genomic adaptation ͉ horizontal gene transfer ͉ phosphorus stress ͉ sulfolipids T he Cuatro Ciénegas Basin (CCB) is located in a valley Ϸ740 m above sea level in the state of Coahuila, Mexico, that measures Ϸ30 km by 40 km and is surrounded by high mountains (Ͼ3,000 m) (Fig. 1). CCB is an enclosed evaporitic basin that receives Ϸ150 mm of annual precipitation. Despite the dry climate of the valley, the CCB harbors an extensive system of springs, streams, and pools (1). The CCB ecosystem is not only characterized by a high endemism of macrooganisms and biodiversity of microorganisms (1, 2), but also by extremely oligotrophic waters that are unable to sustain algal growth, making microbial mats the base of the food web (3). In particular, phosphorus (P) levels in CCB appear to be rather low, because they were below the level of detection of several methods used (0.3 M) and the extremely high biomass C:P and N:P ratios (Ͼ100 by moles) previously reported for CCB stromatolites (3, 4). Unlike the present sea, the Churince spring water is poor in NaCl and carbonates, but it is rich in sulfates, magnesium, and calcium (4). Characterization of the microbiological diversity by sequencing 16S rRNA genes revealed that nearly half of the phylotypes from the CCB were closely related to bacteria from marine environments (2). Bacillus coahuilensis is a free-living, spore-forming bacteria isolated from the water column of a shallow desiccation lagoon in the Churince system at CCB (4) (Fig. 1 A and B). A molecular phylogenetic analysis of 16S rRNA sequences indicates that B. coahuilensis is closely ...

Introduction: The ROS1 gene rearrangement has become an important biomarker in NSCLC. The College of American Pathologists/International Association for the Study of Lung Cancer/Association for Molecular Pathology testing guidelines support the use of ROS1 immunohistochemistry (IHC) as a screening test, followed by confirmation with fluorescence in situ hybridization (FISH) or a molecular test in all positive results. We have evaluated a novel anti-ROS1 IHC antibody (SP384) in a large multicenter series to obtain real-world data.

The aims of this study were to assess the peripapillary retinal nerve fiber layer (RNFL) thickness in patients with Parkinson's disease (PD), to determine its correlation with disease severity, and to define a simple biomarker for predicting clinical severity. One hundred two eyes from 52 patients affected by PD were compared with 97 eyes from 50 age-comparable controls. In all patients, peripapillary RNFL thickness was measured by optical coherence tomography (OCT). We used the Unified Parkinson's Disease Rating Scale (UPDRS) total score and measured responses in the on medication state. Eyes from patients with PD had a statistically significant decrease in average peripapillary RNFL thickness compared with control eyes (P < 0.001). This reduction was observed in every quadrant (inferior, superior, nasal [P < 0.001], and temporal [P = 0.017]) in patients with PD. Furthermore, a strong inverse correlation was found between the PD severity measured according to the UPDRS score and the average peripapillary RNFL thickness (r = -0.615; P < 0.001) and PD duration (r = -0.303; P = 0.002). From these results, we defined a regression equation that predicts the UPDRS score from the above-mentioned variables: UPDRS = 81.6 + 29.6 * log PD duration (years) - 0.6 * RFNL thickness (μm). We observed that, as the evolution and severity of PD progress, the peripapillary RNFL layer thickness, as evaluated by OCT, gradually diminishes. These results suggest that the average peripapillary RNFL thickness measured by OCT might be useful as a biomarker to detect the early onset and progression of PD.