Antimicrobial susceptibility of clinical Helicobacter pylori isolates and its eradication by standard triple therapy: a study in west central region of Colombia.

The aim of the present study was first to isolate Helicobacter pylori from gastric biopsy specimens and to test their antibiotic susceptibility. Second, it was to evaluate the efficacy of the standard triple therapy from patients of the west central region of Colombia. H. pylori positive patients received standard triple therapy with proton pump inhibitor (PPI) (40 mg b.i.d.), clarithromycin (500 mg b.i.d.), and amoxicillin (1 g b.i.d.) for 14 days. Thereafter, antibiotic susceptibility of the isolates was assessed by E-Test. From 94 patients enrolled, 67 were positive for H. pylori by histology or culture. Overall resistance to metronidazole, levofloxacin, rifampicin, clarithromycin, and amoxicillin was 81%, 26.2%, 23.9%, 19%, and 9.5%, respectively. No resistance was found for tetracycline. A total of 54 patients received standard triple therapy, 48 attended follow-ups testing, and of them, 30 had resistance test reports. Overall eradication rate was 81.2%. Second-line treatment was given to eight patients, four of whom were followed up with a 13C urea breath test (UBT) and remained positive for H. pylori. Eradication was significantly higher in patients with clarithromycin susceptible than in resistant strains (95.6% vs 42.8% P = 0.001). The updated percentages of resistance to clarithromycin in this geographical area had increased, so this value must be considered when choosing the treatment regimen.IMPORTANCEAntibiotic resistance in Helicobacter pylori has increased worldwide, as has resistance to multiple antimicrobials (MDRs), which seriously hampers the successful eradication of the infection. The ideal success rate in eradicating H. pylori infection (≥90%) was not achieved in this study (81.2%). This is the first time that MDR is reported (14.3%) in the region; the resistance to clarithromycin increased over time (3.8%-19%), and levofloxacin (26.2%) and rifampicin (23%) resistant isolates were detected for the first time. With these results, strain susceptibility testing is increasingly important, and the selection of treatment regimen should be based on local antibiotic resistance patterns.

Bacillus thuringiensis: A Broader View of Its Biocidal Activity.

Bacillus thuringiensis (Bt) is a Gram-positive bacterium that forms spores and produces parasporal crystalline inclusions containing Cry and Cyt proteins [...].

Relevance of Comet Assay and Phosphorylated-Hsp90α in Cancer Patients' Peripheral Blood Leukocytes as Tools to Assess Cisplatin-based Chemotherapy Clinical Response and Disease Outcome.

Cisplatin (cPt) is a commonly used treatment for solid tumors. The main target of its cytotoxicity is the DNA molecule, which makes the DNA damage response (DDR) crucial for cPt-based chemotherapy. Therefore, it is essential to identify biomarkers that can accurately predict the individual clinical response and prognosis. Our goal was to assess the usefulness of alkaline comet assay and immunocytochemical staining of phosphorylated Hsp90α (p-Hsp90α), γH2AX, and 53BP1 as predictive/prognostic markers. Pre-chemotherapy peripheral blood leukocytes were exposed to cPt in vitro and collected at 0, 24 (T24), and 48 (T48) hr post-drug removal. Healthy subjects were also included. Baseline DNA damage was elevated in cancer patients (variability between individuals was observed). After cPt, patients showed increased γH2AX foci/nucleus (T24 and T48). Both in healthy persons and patients, the nuclear p-Hsp90α and N/C (nuclear/cytoplasmic) ratio augmented (T24), decreasing at T48. Favorable clinical response was associated with high DNA damage and p-Hsp90α N/C ratio following cPt. For the first time, p-Hsp90α significance as a predictive marker is highlighted. Post-cPt-DNA damage was associated with longer disease-free survival and overall survival. Our findings indicate that comet assay and p-Hsp90α (a marker of DDR) would be promising prognostic/predictive tools in cP-treated cancer patients.

Transcriptomics and interactomics during the primary infection of an SfNPV baculovirus on Spodoptera frugiperda larvae.

The fall armyworm (FAW), Spodoptera frugiperda, has been the most devastating pest of corn as well as of other crops in America, and more recently in Africa and Asia. The development of resistance to chemical insecticides led the search for environmentally friendly biological alternatives such as baculoviruses. This study focuses on the primary infection of the baculovirus SfNPV-Ar in the FAW's midgut epithelium, by analyzing the differential expression of transcripts in excised midguts at 6, 12, and 24 h post-infection (hpi), and predicted their interactions. Interaction of viral factors with the infected midgut tissue could alters various cellular processes, such as the apoptotic system due to the up-regulation observed of FABP at 6 hpi and of HSP90 at 24 hpi, along with the down-regulated PRX at 6 hpi and FABP transcripts between 12 and 24 hpi. Changes in transcript regulation could affect the cellular architecture of infected cells due to up-regulation of ARP 2/3 at 6 and 12 hpi, followed by down-regulation at 24 hpi. In relation to protein folding proteins, HSP90 was up-regulated at 24 hpi and PDI was down-regulated between 6 and 12 hpi. With respect to metabolism and cellular transport, AcilBP and ATPS0 were up regulated at 6 hpi and 12 hpi, respectively. In reference to transcription and translation up-regulation of RPL11 at 6 hpi and of FPN32 and RPL19 at 24 hpi was detected, as well as the down-regulation of RPL19 at 6 hpi, of PDI and RPL7 at 12 hpi, and of FABP at 24 hpi. In conclusion, gene regulation induced by viral infection could be related to the cytoskeleton and cellular metabolism as well as to oxidative stress, apoptosis, protein folding, translation, and ribosomal structure. The results presented in this work are an approach to understanding how the virus takes control of the general metabolism of the insect host during the primary infection period.

Examining the impacts of public transit on healthy aging through a natural experiment: study protocols and lessons learned from the Active El Paso project.

This paper describes protocols and experiences from a seven-year natural-experiment study in El Paso, Texas, a border city of predominantly Latino/Hispanic population. The study focuses on how Bus Rapid Transit (BRT) impacts physical activity and thus plays a role in alleviating obesity and related chronic diseases that impact healthy aging. Our protocols describe a longitudinal and case-comparison study, which compared residents exposed to new BRT stations with those who were not. This paper also introduces lessons and experiences to overcome the following challenges: delays in the BRT opening (the main intervention), the COVID-19 pandemic, methodological challenges, participant recruitment and retention, and predatory survey takers. Our transdisciplinary approach was pivotal in addressing these challenges. We also proposed and tested multi-level intervention strategies to reduce modifiable barriers to transit use. Our most important takeaway for researchers, practitioners, and policy makers is the importance of being flexible and ready to adapt to new circumstances. Future natural-experiment researchers need to become more versatile in an increasingly volatile and uncertain world.

Can Entomopathogenic Nematodes and Their Symbiotic Bacteria Suppress Fruit Fly Pests? A Review.

Fruit flies (Diptera: Tephritidae) are serious pests that affect fruit production and marketing. Both third instar larvae and pupae are biological stages that persist in the soil until adult emergence. Entomopathogenic nematodes (ENs) are biological control agents that are used to control agricultural pests in greenhouse or field conditions. Several studies have been carried out under laboratory and field conditions showing how ENs can be applied within an area-wide integrated pest management approach to control fruit fly species in orchards and backyard fruit trees. In this review, we analyze how soil physical characteristics and biotic factors affect the performance of these biological control agents. Of the reviewed papers, more than half evaluated the influence of soil texture, humidity, temperature, and other factors on the performance of infective juveniles (IJs). Abiotic factors that significantly influence the performance of IJs are temperature, humidity, and texture. Among the biotic factors that affect IJs are fungi, bacteria, mites, insects, and earthworms. We conclude that ENs have the potential to be applied in the drip area of fruit trees that are infested by fruit flies and contribute to their suppression. This approach, in conjunction with an area-wide pest management approach, may contribute to pest suppression and increase the sustainability of agroecosystems.

Bacillus thuringiensis: a natural endophytic bacterium found in wild plants.

Despite the fact that Bacillus thuringiensis is the most widely used bacterium in biological pest control, its ecology has been notoriously neglected. Its role in nature is uncertain, and a defined habitat and niche are under discussion. In this report, wild-type strains were isolated from the inner plant tissues as natural endophytic bacteria in wild plants. Once a reliable superficial sterilization technique was standardized, leaf samples from 110 wildlife plant species within 52 families were processed to obtain their endophytic microflora, which were able to grow in artificial media. From 93 morphologically different isolates, 22 showed the typical sporangium morphology of B. thuringiensis (endospore and parasporal bodies). These isolates were identified and characterized by their 16S ribosomal RNA, hag gene, MLST, and cry gene sequences. Also, isolates were characterized by Bc-RepPCR and parasporal body protein content. All the isolates showed at least some of the typical B. thuringiensis features tested, but 10 showed information in all those features, which, in a rigorous selection, were taken as B. thuringiensis sensu stricto strains. Only three subspecies were identified: five kurstaki, four nigeriensis, and one thuringiensis. None showed toxicity against mosquito larvae or Caenorhabditis elegans, and only one showed significant toxicity against Manduca sexta larvae. The role of B. thuringiensis as a natural endophytic bacterium is discussed.

A novel hydro-pneumatic fluid percussion device for inducing traumatic brain injury: assessment of sensory, motor, cognitive, molecular, and morphological outcomes in rodents.

Introduction: The fluid percussion method is widely used to induce brain injury in rodents. However, this approach has several limitations, including variability in the resulting damage, which is attributed to factors such as manual control of the mass used to generate the desired pressure. To address these issues, several modifications to the original method have been proposed. Methods: In this study, we present a novel device called the Hydro-pneumatic Fluid Percussion Device, which delivers fluid directly to a lateral region of the brain to induce injury. To validate this model, three groups of male and female rats were subjected to lateral fluid percussion using our device, and the resulting damage was evaluated using sensory, motor, and cognitive tests, measurements of serum injury biomarkers, and morphological analysis via cresyl violet staining. Results: Our results demonstrate that this new approach induced significant alterations in all parameters evaluated. Discussion: This novel device for inducing TBI may be a valuable alternative for modeling brain injury and studying its consequences.

Acute cholecystitis complicated with gallbladder empyema due to Mycobacterium tuberculosis in a patient with diabetes mellitus: a case report.

Acute cholecystitis severity ranges from mild to very severe, and its most dreadful complication is gallbladder empyema. It can be caused by several etiologic agents, but Mycobacterium tuberculosis is not common among them. Here we present a 61-year-old female who lives in an area of high tuberculosis endemicity and has type 2 diabetes mellitus. She came to our hospital with a 2-day history of moderate-to-severe colicky right upper quadrant abdominal pain and other clinical manifestations compatible with AC. Imaging studies confirmed the diagnosis. An emergency open cholecystectomy was performed and the gallbladder was sent for histopathologic examination. M. tuberculosis was identified by molecular studies and the treatment was adjusted. The patient recovered uneventfully. The clinical history and physical examination are essential for raising the index of suspicion, but complementary evaluation with imaging studies is necessary to confirm the diagnosis and evaluate its complications. Tuberculosis is a major health problem worldwide, and health professionals should be aware of its clinical spectrum to approach and manage common and uncommon presentations within their scope of attention.

Biological, morphological, and molecular characterization of the baculovirus PlxyMNPV_LBIV-11, and its virulence towards Plutella xylostella, Trichoplusia ni, and Spodoptera frugiperda larvae.

PlxyMNPV_LBIV-11 is an alphabaculovirus strain, isolated from Plutella xylostella larvae. This work characterized this strain at a biological, morphological, and molecular level to evaluate its similarity with other baculoviruses. Its ultrastructure showed a multiple arrangement of nucleocapsids within enveloped virions, all occluded within large cubical polyhedra. PlxyMNPV_LBIV-11 showed infectivity on the Hi5 and Sf9 cell lines, despite these being from heterologous origin. This in vitro infectivity was observed using either BVs or by transfection with genomic DNA. Restriction fragment patterns of PlxyMNPV_LBIV-11, using the enzymes EcoRI, BamHI and HindIII, showed a high relationship with those patterns shown by AcMNPV, except for one or two differential bands with each enzyme. Sequences of core genes lef-8 and lef-9 and the conserved polh gene showed identities ranging from 98 to 100% when compared with those of AcMNPV. Somewhat lower was the sequence identity of the gp64 gene (94%) as compared with those of AcMNPV and PlxyMNPV_CL3, which might be related to the difference in virulence. Besides, the presence of this gene in PlxyMNPV_LBIV-11 indicates that it belongs to group 1 of alphabaculoviruses. A phylogram was estimated with the core and conserved gene sequences, corroborating its high relationship with AcMNPV and PlxyMNPV_CL3. Bioassays were performed with P. xylostella larvae reared on a meridic diet, whose LC50 values indicated lower virulence than AcMNPV when tested against P. xylostella, Spodoptera frugiperda, and Trichoplusia ni larvae. Its virulence against S. frugiperda was only seven times lower than AcMNPV. Its potential as a biological control agent is discussed.

Molecular and Toxicological Characterization of a Bacillus thuringiensis Strain Expressing a Vip3 Protein Highly Toxic to Spodoptera frugiperda (Lepidoptera: Noctuidae).

The characterization of the Bacillus thuringiensis (Berliner) LBIT-418 strain was based on a previous work which indicated its high insecticidal potential. Therefore, toxicological, molecular, and biochemical characterizations were conducted in this work to identify its unique features and its potential to be developed as a bioinsecticide. This strain, originally isolated from a healthy mosquito larva, was identified within the subspecies kenyae by sequencing of the hag gene and by the multilocus sequence typing (MLST) technique. Genes cry1Ac2, cry1Ea3, cry2Aa1 and cry2Ab4, and a cry1Ia were detected in its genome, in addition to a vip3Aa gene. In this research, the latter protein was successfully cloned, expressed, and purified and showed high toxicity towards the fall armyworm, Spodoptera frugiperda (J.E. Smith), fourth instar larvae in bioassays using the microdroplet ingestion technique, estimating an LD50 of 21.38 ng/larva. Additional bioassays were performed using the diet surface inoculation technique of the strain's spore-crystal complex against diamondback moth larvae, Plutella xylostella (Linnaeus), estimating an LC50 of 10.22 ng/cm2. Its inability to produce ß-exotoxin was demonstrated by bioassays against the nematode Caenorhabditis elegans Maupas and by HPLC analysis. These results support the high potential of this strain to be developed as a bioinsecticide.

DNA Damage Repair Proteins, HSP27, and Phosphorylated-HSP90α as Predictive/Prognostic Biomarkers of Platinum-based Cancer Chemotherapy: An Exploratory Study.

Platinum analogs are commonly used for cancer treatment. There is increasing interest in finding biomarkers which could predict and overcome resistance, because to date there is no reliable predictive/prognostic marker for these compounds. Here we studied the immunohistochemical expression of proteins involved in DNA damage response and repair (γH2AX, 53BP1, ERCC1, MLH1, and MSH2) in primary tumor tissues from patients treated with platinum-based chemotherapy. Levels and localization of Heat Shock Protein (HSP)27 and phospho-(Thr5/7)-HSP90α (p-HSP90α) were also determined. The implications in clinical response, disease-free survival and overall survival were analyzed. High γH2AX and 53BP1 expressions were associated with poor clinical response. Nuclear p-HSP90α, as well as nuclear absence and low cytoplasmic expression of HSP27 correlated with good response. Patients with high γH2AX and high cytoplasmic HSP27 expressions had shorter overall survival and disease-free survival. MLH1, MSH2, or ERCC1 were not associated with clinical response or survival. We report the potential utility of p-HSP90α, HSP27, γH2AX, and 53BP1 as predictive/prognostic markers for platinum-based chemotherapy. We present the first study that evaluates the predictive and prognostic value of p-HSP90α in primary tumors. Our research opens new possibilities for clinical oncology and shows the usefulness of immunohistochemistry for predicting chemotherapy response and prognosis in cancer.

Comparación de la fuerza de adhesión a dentina de cementos de autograbado vs grabado total / Comparison of bond strength to dentin of self-etching cements vs total etching

Los sistemas cementantes han mejorado notablemente, los objetivos que persiguen los nuevos cementos es que la adhesión sea duradera y conseguir siempre que sea posible una interface cerrada con un sellado perfecto. Se han podido desarrollar nuevas técnicas y nuevos materiales de cementación que han ido perfeccionando la unión del material restaurador al diente. En el presente estudio se compara la fuerza de adhesión a dentina de cementos de autograbado y cementos de grabado total para comprobar los efectos positivos en el grabado de la dentina. Para el estudio se utilizaron dos cementos a base de resina (RelyX U200 Clicker 3M y RelyX Ultimate 3M). Se encapsularon 40 molares en acrílico en dos grupos de 20 muestras para la aplicación de dos sistemas cementantes de autograbado (grupo 1) y de grabado total (grupo 2), respectivamente, se desgastaron hasta descubrir la dentina; siguiendo las especificaciones del fabricante se colocó el cemento en cada grupo, y después se sometieron a pruebas de cizalla en una máquina de ensayo universal Instron. La medida expresada en megapascales (MPa) fue: grupo 1 = 7.5569 y grupo 2 = 12.6444. En este caso fueron analizados dos grupos, tomándose la primera significancia bilateral. Se realiza la prueba en t de Student, con 95% de intervalo de confianza en la diferencia, demostrando así que el cemento RelyX Ultimate 3M tiene mayor fuerza de adhesión que el cemento RelyX U200 Clicker 3M. Nuestra investigación fue factible y llegamos a nuestro propósito, en el cual comprobamos la mayor adhesión de cementos de grabado total, los cuales son sometidos a un previo tratamiento del diente (AU)

Virulence and genetic characterization of six baculovirus strains isolated from different populations of Spodoptera frugiperda (Lepidoptera: Noctuidae).

Fall armyworm (FAW), Spodoptera frugiperda (Smith, 1797), is a polyphagous, voracious, and economically important agricultural pest. Biological control of FAW is a strategy that must be further explored. This study evaluated six baculovirus strains isolated from infected FAW larvae from Mexico, Argentina, Honduras, and the United States. Five alphabaculoviruses (SfNPV-An2, SfNPV-Arg, SfNPV-Fx, SfNPV-Ho, and SfNPV-Sin) and one betabaculovirus (SfGV-RV) were tested against FAW larvae, showing a wide diversity of virulence levels among strains when their estimated LC50s were compared, being SfNPV-Arg, SfNPV-Ho and SfNPV-Fx more virulent than SfNPV-An2, SfNPV-Sin, and SfGV-RV. To determine any virulence difference in vitro studies of these isolates, Sf9 cell cultures were used. Interestingly, only ODVs from four of the test SfNPV strains showed infectivity on Sf9 cell cultures, and some differences in virulence were observed. Genomic restriction analyses and partial sequences of lef-8, lef-9, and polh/granulin genes showed little variability among alphabaculoviruses, both, among them and with previously reported sequences. However, sequences from SfGV-RV were closer to previously reported sequences from the SfGV-VG008 strain than the SfGV-Arg and SfGV-VG014 strains. The great difference in the in vivo virulence was not correlated with great similarity among the isolates. The characterization of these six baculovirus isolates offers the basis for exploring their potential as biological control agents against S. frugiperda, as well the initial studies on their specific infection mechanisms, evolution, and ecology.

Coffea arabica L. Resistant to Coffee Berry Borer (Hypothenemus hampei) Mediated by Expression of the Bacillus thuringiensis Cry10Aa Protein.

Coffea spp. are tropical plants used for brewing beverages from roasted and grounded seeds, the favorite drink in the world. It is the most important commercial crop plant and the second most valuable international commodity after oil. Global coffee trade relies on two Coffea species: C. arabica L. (arabica coffee) comprising 60% and C. canephora (robusta) comprising the remaining 40%. Arabica coffee has lower productivity and better market price than robusta. Arabica coffee is threatened by disease (i.e., coffee leaf rust), pests [i.e., Hypothenemus hampei or coffee berry borer (CBB) and nematodes], and susceptibility to climate change (i.e., drought and aluminum toxicity). Plant biotechnology by means of tissue culture inducing somatic embryogenesis (SE) process, genetic transformation, and genome editing are tools that can help to solve, at least partially, these problems. This work is the continuation of a protocol developed for stable genetic transformation and successful plant regeneration of arabica coffee trees expressing the Bacillus thuringiensis (Bt) toxin Cry10Aa to induce CBB resistance. A highly SE line with a high rate of cell division and conversion to plants with 8-month plant regeneration period was produced. To validate this capability, gene expression analysis of master regulators of SE, such as BABY BOOM (BBM), FUS3, and LEC1, embryo development, such as EMB2757, and cell cycle progression, such as ETG1 and MCM4, were analyzed during induction and propagation of non-competent and highly competent embryogenic lines. The particle bombardment technique was used to generate stable transgenic lines after 3 months under selection using hygromycin as selectable marker, and 1 month in plant regeneration. Transgenic trees developed fruits after 2 years and demonstrated expression of the Bt toxin ranging from 3.25 to 13.88 µg/g fresh tissue. Bioassays with transgenic fruits on CBB first instar larvae and adults induced mortalities between 85 and 100% after 10 days. In addition, transgenic fruits showed a seed damage lower than 9% compared to 100% of control fruits and adult mortality. This is the first report on stable transformation and expression of the Cry10Aa protein in coffee plants with the potential to control CBB.

Identification and characterization of a new cry-like gene found in a Bacillus cereus strain.

Bacillus thuringiensis is the most successful microbial insecticide against different pests in agriculture and vectors of diseases. Its activity is mostly attributed to the Cry proteins expressed during its sporulation phase. However, these proteins are not exclusive to B. thuringiensis. Some cry genes have been found in other Bacillus species, or even in other genera. In this work, cry genes were searched in 223 acrystalliferous bacillaceous strains. From these strains 13 amplicons were obtained, cloned, and sequenced; however, only 6 amplicons tested positive for cry-like genes, and the 6 isolates showed to be the same strain. We report the characterization of an unusual strain of B. cereus (LBIC-004) which is unable to form protein inclusions during the sporulation phase. LBIC-004 showed a high identity to B. cereus using the sequences of 16S rRNA, gyrB and hag genes; in addition, a unique plasmid pattern of the strain was obtained. A 1953-bp cry gene was identified, coding for a 651 amino acid protein with a molecular weight of 74.9 kDa. This protein showed a predicted three-domain structure, similar to all Cry proteins. However, the amino acid sequence of the protein showed only 41% identity its highest hit: the Cry8Ca1 protein, indicating the uniqueness of this cry-like gene. It was cloned and transferred into a mutant acrystalliferous B. thuringiensis strain which was used in bioassays against Caenorhabditis elegans, Aedes aegypti, Manduca sexta and Phyllophaga sp. The recombinant strain showed no crystal formation and no toxicity to the tested species.

Isolation and characterization of two highly insecticidal, endophytic strains of Bacillus thuringiensis.

Recent discovery of endophytic strains of Bacillus thuringiensis significantly improves the knowledge on its ecology. It also may be a new source for the isolation of insecticidal strains. This report shows the characterization of two endophytic, highly insecticidal strains of B. thuringiensis. Strains LBIT-1250L and LBIT-1251P were isolated from lavender and Poinsettia sap, respectively. Their parasporal crystals were very similar in morphology to those shown by serotypes israelensis and kurstaki, respectively. Bioassays on Aedes aegypti fourth instar larvae and on Manduca sexta first instar larvae, respectively, showed significantly higher levels of toxicity than those of their standard counterparts, IPS-82 (israelensis) and HD-1 (kurstaki) strains, respectively. Characterization of both strains included the sequencing of flagellin (hag) gene, plasmid and Bc Rep-PCR patterns and crystal protein content. All four characterization features indicated that LBIT1250L is highly related to the IPS-82 standard (serotype H-14: israelensis); while the LBIT-1251P was highly related to the HD-1 standard (serotype H-3a3b3c kurstaki). These results indicate that endophytic strains of B. thuringiensis may be a new source of potential insecticidal strains and opens more in-depth studies about the role of this bacterium in such a specialized habitat.

Osmotic stress-induced somatic embryo maturation of coffee Coffea arabica L., shoot and root apical meristems development and robustness.

Somatic embryogenesis (SE) is the most important plant biotechnology process for plant regeneration, propagation, genetic transformation and genome editing of coffee, Coffea arabica L. Somatic embryo (SEs) conversion to plantlets is the principal bottleneck for basic and applied use of this process. In this study we focus on the maturation of SEs of C. arabica var. Typica. SEs conversion to plantlet up to 95.9% was achieved under osmotic stress, using 9 g/L gelrite, as compared with only 39.34% in non-osmotic stress. Mature SEs induced in osmotic stress developed shoot and root apical meristems, while untreated SEs were unable to do it. C. arabica regenerated plants from osmotic stress were robust, with higher leaf and root area and internode length. To understand a possible regulatory mechanism, gene expression of key genes of C. arabica, homologous to sequences in the Arabidopsis thaliana genome, were analyzed. A set of two component system and cytokinin signaling-related coding genes (AHK1, AHK3, AHP4 and ARR1) which interact with WUSCHEL and WOX5 homedomains and morphogenic genes, BABY-BOOM, LEC1, FUS3 and AGL15, underwent significant changes during maturation of SEs of C. arabica var. Typica. This protocol is currently being applied in genetic transformation with high rate of success.

Nano dot blot: An alternative technique for protein identification and quantification in a high throughput format.

BACKGROUND: Dot blot technique has been used in a similar way to western blotting, with the major difference being the lack of protein separation with electrophoresis. Protein samples are spotted over a membrane paper, the identification and quantification of a protein is achieved by immunodetection procedures such as colorimetry, fluorescence or chemiluminescence. This technique is widely accepted, but it uses large amounts of sample and antibodies to reveal the presence of the target protein. Significant milestones have been reached to achieve better results with the use of less sample and reagents; however, the ninety-six-well format is still in use. NEW METHOD: In this work, we propose an innovation to this technique, reducing the amount of sample and antibodies to identify a specific protein when compared to the regular dot blot method. Procedure consists of using a sample volume of approximately 200 nanoliters deposited with a multineedle device developed by our group. RESULTS: Five samples of standard protein or antigen can be spotted in a Cartesian format to identify and quantify the protein involved in physiological or pathological conditions. In addition, at least five replicates of sample or antigen are used to enable better statistics to calculate the concentration of every standard and the protein present in a sample. CONCLUSIONS: Hundreds of samples can be deposited in a few minutes and analyzed in a single experimental session. To validate this method, which we called nano dot blot, six proteins involved in the inflammation process were tested in acute and chronic rat models of seizures.

Selection and characterization of two Bacillus thuringiensis strains showing nematicidal activity against Caenorhabditis elegans and Meloidogyne incognita.

Bacillus thuringiensis has been widely used as a biological control agent against insect pests. Additionally, nematicidal strains have been under investigation. In this report, 310 native strains of B. thuringiensis against Caenorhabditis elegans were tested. Only the LBIT-596 and LBIT-107 strains showed significant mortality. LC50s of spore-crystal complexes were estimated at 37.18 and 31.89 µg/mL for LBIT-596 and LBIT-107 strains, respectively, while LC50s of partially purified crystals was estimated at 23.76 and 20.25 µg/mL for LBIT-596 and LBIT-107, respectively. The flagellin gene sequence and plasmid patterns indicated that LBIT-596 and LBIT-107 are not related to each other. Sequences from internal regions of a cry5B and a cyt1A genes were found in the LBIT-596 strain, while a cry21A, a cry14A and a cyt1A genes were found in the LBIT-107 strain. Genome sequence of the LBIT-107 strain showed new cry genes, along with other virulence factors, hence, total nematicidal activity of the LBIT-107 strain may be the result of a multifactorial effect. The highlight of this contribution is that translocation of spore-crystal suspensions of LBIT-107 into tomato plants inoculated at their rhizosphere decreased up to 90% the number of galls of Meloidogyne incognita, perhaps the most important nematode pest in the world.