Preserving US microbe collections sparks future discoveries.

Collections of micro-organisms are a crucial element of life science research infrastructure but are vulnerable to loss and damage caused by natural or man-made disasters, the untimely death or retirement of personnel, or the loss of research funding. Preservation of biological collections has risen in priority due to a new appreciation for discoveries linked to preserved specimens, emerging hurdles to international collecting and decreased funding for new collecting. While many historic collections have been lost, several have been preserved, some with dramatic rescue stories. Rescued microbes have been used for discoveries in areas of health, biotechnology and basic life science. Suggestions for long-term planning for microbial stocks are listed, as well as inducements for long-term preservation.

Biolog Phenotype Microarray Is a Tool for the Identification of Multidrug Resistance Efflux Pump Inducers.

Multidrug resistance efflux pumps frequently present low levels of basal expression. However, antibiotic-resistant mutants that overexpress these resistance determinants are selected during infection. In addition, increased expression of efflux pumps can be induced by environmental signals/cues, which can lead to situations of transient antibiotic resistance. In this study, we have applied a novel high-throughput methodology in order to identify inducers able to trigger the expression of the Stenotrophomonas maltophilia SmeVWX and SmeYZ efflux pumps. To that end, bioreporters in which the expression of the yellow fluorescent protein (YFP) is linked to the activity of either smeVWX or smeYZ promoters were developed and used for the screening of potential inducers of the expression of these efflux pumps using Biolog phenotype microarrays. YFP production was also measured by flow cytometry, and the levels of expression of smeV and smeY in the presence of a set of selected compounds were also determined by real-time reverse transcription-PCR (RT-PCR). The expression of smeVWX was induced by iodoacetate, clioquinol, and selenite, while boric acid, erythromycin, chloramphenicol, and lincomycin triggered smeYZ expression. The susceptibility to antibiotics that are known substrates of the efflux pumps decreased in the presence of the inducers. However, the analyzed multidrug efflux systems did not contribute to S. maltophilia resistance to the studied inducers. To sum up, the use of fluorescent bioreporters in combination with Biolog plates is a valuable tool for identifying inducers of the expression of bacterial multidrug resistance efflux pumps, and likely of other bacterial systems whose expression is regulated in response to signals/cues.

The effect of feed composition on anaerobic co-digestion of animal-processing by-products.

Four streams and their mixtures have been considered for anaerobic co-digestion, all of them generated during pig carcasses processing or in related industrial activities: meat flour (MF), process water (PW), pig manure (PM) and glycerin (GL). Biochemical methane potential assays were conducted at 37 °C to evaluate the effects of the substrate mix ratio on methane generation and process behavior. The results show that the co-digestion of these products favors the anaerobic fermentation process when limiting the amount of meat flour in the mixture to co-digest, which should not exceed 10%. The ratio of other tested substrates is less critical, because different mixtures reach similar values of methane generation. The presence in the mixture of process water contributes to a quick start of the digester, something very interesting when operating an industrial reactor. The analysis of the fraction digested reveals that the four analyzed streams can be, a priori, suitable for agronomic valorization once digested.

'Timed up and go' and brain atrophy: a preliminary MRI study to assess functional mobility performance in multiple sclerosis.

Motor and cognitive disabilities are related to brain atrophy in multiple sclerosis (MS). 'Timed up and go' (TUG) has been recently tested in MS as functional mobility test, as it is able to evaluate ambulation/coordination-related tasks, as well as cognitive function related to mobility. The objective of this study is to evaluate the relationship between brain volumes and TUG performances. Inclusion criteria were a diagnosis of MS and the ability to walk at least 20 m. TUG was performed using a wearable inertial sensor. Times and velocities of TUG sub-phases were calculated by processing trunk acceleration data. Patients underwent to a brain MRI, and volumes of whole brain, white matter (WM), grey matter (GM), and cortical GM (C) were estimated with SIENAX. Sixty patients were enrolled. Mean age was 41.5 ± 11.6 years and mean EDSS 2.3 ± 1.2. Total TUG duration was correlated to lower WM (ρ = 0.358, p = 0.005) and GM (ρ = 0.309, p = 0.017) volumes. A stronger association with lower GM volume was observed for intermediate (ρ = 0.427, p = 0.001) and final turning (ρ = 0.390, p = 0.002). TUG is a useful tool in a clinical setting as it can not only evaluate patients' disability in terms of impaired functional mobility, but also estimate pathological features, such as grey atrophy.

Vitamin K3 Induces the Expression of the Stenotrophomonas maltophilia SmeVWX Multidrug Efflux Pump.

Stenotrophomonas maltophilia is an opportunistic pathogen with increasing prevalence, which is able to cause infections in immunocompromised patients or in those with a previous pathology. The treatment of the infections caused by this bacterium is often complicated due to the several intrinsic antibiotic resistance mechanisms that it presents. Multidrug efflux pumps are among the best-studied mechanisms of S. maltophilia antibiotic resistance. Some of these efflux pumps have a basal expression level but, in general, their expression is often low and only reaches high levels when the local regulator is mutated or bacteria are in the presence of an effector. In the current work, we have developed a yellow fluorescent protein (YFP)-based sensor with the aim to identify effectors able to trigger the expression of SmeVWX, an efflux pump that confers resistance to quinolones, chloramphenicol, and tetracycline when it is expressed at high levels. With this purpose in mind, we tested a variety of different compounds and analyzed the fluorescence signal given by the expression of YFP under the control of the smeVWX promoter. Among the tested compounds, vitamin K3, which is a compound belonging to the 2-methyl-1,4-naphthoquinone family, is produced by plants in defense against infection, and has increasing importance in human therapy, was able to induce the expression of the SmeVWX efflux pump. In addition, a decrease in the susceptibility of S. maltophilia to ofloxacin and chloramphenicol was observed in the presence of vitamin K3, in both wild-type and smeW-deficient strains.

Walking improvements with nabiximols in patients with multiple sclerosis.

Recently, nabiximols was approved as a treatment in MS spasticity. Data leading to approval and clinical use of nabiximols, although widely recognised, are based on subjective scales. Movement analysis procedures would obtain more detailed data about the impact on mobility. The aim of the study was to quantitatively assess the functional modification of gait patterns induced by nabiximols in MS. We evaluated three-dimensional gait analysis (spatial-temporal and kinematic) variation by means of one-way ANOVA. Twenty patients were enrolled-9 male and 11 female-with mean EDSS of 5.3 (SD ± 0.81) and mean reduction of numerical rating scale during nabiximols treatment of 1.88. The spatial-temporal parameters of gait revealed an increased speed (+15 %, p < 0.001), cadence (+6 %, p < 0.001) and stride length (+10 %, p < 0.001) after treatment. Regarding the kinematics data, the Gait Profile Score after treatment was reduced by 10 % (p < 0.001): Significant changes involved the pelvic area, hip rotation and knee flexion-extension. We found that nabiximols is able to improve the speed, cadence and stride length. Furthermore, the dynamics of the proximal segment of the legs and the knee movement results after treatment are closer to the physiologic values.

[Osteomyelitis: a probable, uncommon etiology agent]. / Osteomielite: un probabile insolito agente eziologico.

The relation of infectious agents to arthritis is an area of great interest to the rheumatologist. Septic arthritis of bacterial origin accounts for approximately 6.5% of all childhood arthritides. Septic arthritis usually results from haematogenous spread from a focus of infection elsewhere in the body, but also by direct extension of an infection from overlying soft tissues or bone or traumatic invasion of the joint. As a result, if a focus of underlying osteomyelitis breaks throught the metaphysis, it may enter the joint and result in septic arthritis. Systemic signs of illness are fever, severe bone pain, and tenderness with or without local swelling. A wide range of microorganism can cause septic arthritis in children; Staphylococcus aureus and nongroup A and B streptococci are most common overall. However, different organisms are more common at some ages and in certain circumstances. Kingella kingae is an emerging pathogen in young children under 4 years of age. The clinical presentation of K. kingae invasive infection is often subtle and may be associated to mild to moderate biologic inflammatory responses. Affected children often have few signs and symptoms of osteoarticular infections. Early MRI is useful in differentiating K kingae from Gram-positive cocci in osteoarticular infections. Cartilaginous involvement, modest soft tissue and bone reaction suggest K. kingae. It's very important to include K. kingae in differential diagnosis of osteoarticular infections in young children. We report an unusual case of osteomyelitis: clinical manifestations and MRI are suggestive for K kingae infection.

Muscle ischemic preconditioning does not improve performance during self-paced exercise.

Muscle ischemic preconditioning (IP) has been found to improve exercise performance in laboratory tests. This investigation aims at verifying whether performance is improved by IP during self-paced exercise (SPE) in the field. 11 well-trained male runners performed 3 randomly assigned 5 000 m self-paced running tests on an outdoor track. One was the reference (RT) test, while the others were performed following muscle IP (IPT) and a control sham test (ST). Average speeds were measured during each test. Mean values in oxygen uptake (VO2), aerobic energy cost (AEC) during race and post-race blood lactate (BLa) were gathered. Data showed that none of the studied variables were affected by IPT or ST with respect to the RT test. Average speeds were 4.63±0.31, 4.62±0.31 and 4.60±0.25 m·s(-1) for the RT, the ST and the IPT tests, respectively. Moreover, there was no difference among tests in speed reached during each lap. VO2 was 3.5±0.69, 3.74±0.85 and 3.62±1.19 l·min(-1). AEC was 1.04±0.15, 1.08±0.1 and 1.09±0.15 kcal·kg(-1)·km(-1). Finally, post-race BLa levels reached 12.85±3.54, 11.88±4.74 and 12.82±3.6 mmol·l(-1). These findings indicate that performance during SPE is not ameliorated by ischemic preconditioning, thereby indicating that IP is not suitable as an ergogenic aid.

Family directed umbilical cord blood banking for acute leukemia: usage rate in hematopoietic stem cell transplantation.

Family-directed umbilical cord blood (UCB) collection and banking is indicated in women delivering healthy babies who already have a member of their own family with a disease potentially treatable with an allogeneic hematopoietic stem cell (HSCs) transplantation (HSCT). The rapid availability of UCB is an important issue in HSCs procurement particularly for recipients with acute leukemia who urgently need HSCT. The aims of this study were to assess the usage rate of family UCB collections directed to patients with acute leukemia and to investigate the factors influencing the usage rate. A total of 113 families were enrolled, 118 UCB units were successfully collected and one collection failed due to emergency occurred during delivery. Among these, 7 collections were required for children who were in urgent need of a transplant: three HLA-matched units were successfully transplanted, respectively after 2, 5 and 6 months from collection; three collections resulted HLA-mismatched, while HLA-typing is pending for one unit. The remaining collections were mostly required for potential future use, among these units only one was transplanted in a HLA compatible sibling after 3 years and 4 months from collection. After a median time of storage of 8.5 years (range 0.1-20 years) a total of 4/118 (3.4 %) collection has been transplanted. During this time interval, considering only patients who have had the need of a transplant, the main factor influencing low utilization rate of UCB collections was due to HLA disparity, indeed among typed UCB unit mostly (77 %) resulted HLA mismatched with the intended recipient.

Primer modification improves rapid and sensitive in vitro and field-deployable assays for detection of high plains virus variants.

A high consequence pathogen, High plains virus (HPV) causes considerable damage to wheat if the crop is infected during early stages of development. Methods for the early, accurate, and sensitive detection of HPV in plant tissues are needed for the management of disease outbreaks and reservoir hosts. In this study, the effectiveness of five methods-real-time SYBR green and TaqMan reverse transcription-quantitative PCR (RT-qPCR), endpoint RT-PCR, RT-helicase dependent amplification (RT-HDA) and the Razor Ex BioDetection System (Razor Ex)-for the broad-range detection of HPV variants was evaluated. Specific PCR primer sets and probes were designed to target the HPV nucleoprotein gene. Primer set HPV6F and HPV4R, which amplifies a product of 96 bp, was validated in silico against published sequences and in vitro against an inclusivity panel of infected plant samples and an exclusivity panel of near-neighbor viruses. The primers were modified by adding a customized 22 nucleotide long tail at the 5' terminus, raising the primers' melting temperature (Tm; ca. 10°C) to make them compatible with RT-HDA (required optimal Tm = 68°C), in which the use of primers lacking such tails gave no amplification. All of the methods allowed the detection of as little as 1 fg of either plasmid DNA carrying the target gene sequence or of infected plant samples. The described in vitro and in-field assays are accurate, rapid, sensitive, and useful for pathogen detection and disease diagnosis, microbial quantification, and certification and breeding programs, as well as for biosecurity and microbial forensics applications.

Highly Sensitive End-Point PCR and SYBR Green qPCR Detection of Phymatotrichopsis omnivora, Causal Fungus of Cotton Root Rot.

Phymatotrichopsis omnivora, the causal pathogen of cotton root rot, is a devastating ascomycete that affects numerous important dicotyledonous plants grown in the southwestern United States and northern Mexico. P. omnivora is notoriously difficult to isolate from infected plants; therefore methods for accurate and sensitive detection directly from symptomatic and asymptomatic plant samples are needed for disease diagnostics and pathogen identification. Primers were designed for P. omnivora based on consensus sequences of the nuclear ribosomal internal transcribed spacer (ITS) region of geographically representative isolates. Primers were compared against published P. omnivora sequences and validated against DNA from P. omnivora isolates and infected plant samples. The primer combinations amplified products from a range of P. omnivora isolates representative of known ITS haplotypes using standard end-point polymerase chain reaction (PCR) methodology. The assays detected P. omnivora from infected root samples of cotton (Gossypium hirsutum) and alfalfa (Medicago sativa). Healthy plants and other relevant root pathogens did not produce PCR products with the P. omnivora-specific primers. Primer pair PO2F/PO2R was the most sensitive in end-point PCR assays and is recommended for use for pathogen identification from mycelial tissue and infected plant materials when quantitative PCR (qPCR) is not available. Primer pair PO3F/PO2R was highly sensitive (1 fg) when used in SYBR Green qPCR assays and is recommended for screening of plant materials potentially infected by P. omnivora or samples with suboptimal DNA quality. The described PCR-based detection methods will be useful for rapid and sensitive screening of infected plants in diagnostic laboratories, plant health inspections, and plant breeding programs.

Development of a rapid, sensitive, and field-deployable razor ex BioDetection system and quantitative PCR assay for detection of Phymatotrichopsis omnivora using multiple gene targets.

A validated, multigene-based method using real-time quantitative PCR (qPCR) and the Razor Ex BioDetection system was developed for detection of Phymatotrichopsis omnivora. This soilborne fungus causes Phymatotrichopsis root rot of cotton, alfalfa, and other dicot crops in the southwestern United States and northern Mexico, leading to significant crop losses and limiting the range of crops that can be grown in soils where the fungus is established. It is on multiple lists of regulated organisms. Because P. omnivora is difficult to isolate, accurate and sensitive culture-independent diagnostic tools are needed to confirm infections by this fungus. Specific PCR primers and probes were designed based on P. omnivora nucleotide sequences of the genes encoding rRNA internal transcribed spacers, beta-tubulin, and the second-largest subunit of RNA polymerase II (RPB2). PCR products were cloned and sequenced to confirm their identity. All primer sets allowed early detection of P. omnivora in infected but asymptomatic plants. A modified rapid DNA purification method, which facilitates a quick (∼30-min) on-site assay capability for P. omnivora detection, was developed. Combined use of three target genes increased the assay accuracy and broadened the range of detection. To our knowledge, this is the first report of a multigene-based, field-deployable, rapid, and reliable identification method for a fungal plant pathogen and should serve as a model for the development of field-deployable assays of other phytopathogens.

Comparative assessment of 5' A/T-rich overhang sequences with optimal and sub-optimal primers to increase PCR yields and sensitivity.

Efficient PCR amplifications require precisely designed and optimized oligonucleotide primers, components, and cycling conditions. Despite recent software development and reaction improvement, primer design can still be enhanced. The aims of this research are to understand (1) the effect on PCR efficiency and DNA yields of primer thermodynamics parameters, and (2) the incorporation of 5' A/T-rich overhanging sequences (flaps) during primer design. Two primer sets, one optimal (ΔG = 0) and one sub-optimal (ΔG = 0.9), were designed using web interface software Primer3, BLASTn, and mFold to target a movement protein gene of Tobacco mosaic virus. The optimal primer set amplifies a product of 195 bp and supports higher PCR sensitivity and yields compared to the sub-optimal primer set, which amplifies a product of 192 bp. Greater fluorescence was obtained using optimal primers compared to that with sub-optimal primers. Primers designed with sub-optimal thermodynamics can be substantially improved by adding 5' flaps. Results indicate that even if the performance of some primers can be improved substantially by 5' flap addition, not all primers will be similarly improved. Optimal 5' flap sequences are dependent on the primer sequences, and alter the primer's T m value. The manipulation of this feature may enhance primer's efficiency to increase the PCR sensitivity and DNA yield.

Laparoscopic versus open appendectomy for the treatment of complicated appendicitis.

AIM: Laparoscopic Appendectomy (LA) is widely performed for the treatment of acute appendicitis. However the use of laparoscopic approach for complicated appendicitis is controversial, in particular because it has been reported an increased risk of postoperative IntraAbdominal Abscess (IAA). The aim of this study was to compare the outcomes of LA versus Open Appendectomy (OA) in the treatment of complicated appendicitis, especially with regard to the incidence of postoperative IAA. PATIENTS AND METHODS: A retrospective study of all patients treated at our institution for complicated appendicitis, from May 2004 to June 2009, was performed. Data collection included demographic characteristics, postoperative complications, conversion rate, and length of hospital stay. RESULTS: Thirty-eight patients with complicated appendicitis were analysed. Among these, 18 (47,3%) had LA and 20 (52,7%) had OA. There were no statistical differences in characteristics between the two groups. The incidence of postoperative IAA was higher (16,6%), although not statistically significant, in the LA compared with OA group (5%). On the other hand the rate of wound infection was lower (5%) in the LA versus OA (20%). CONCLUSION: Our study indicated that LA should be utilised with caution in case of perforated appendicitis, because it is associated with an increased risk of postoperative IAA compared with OA.

Nitrate estimation in the denitrifying post-filtration unit of a municipal wastewater treatment plant: the Viikinmäki case.

In this work we present and discuss the design of an array of soft-sensors to estimate the nitrate concentration in the denitrifying post-filtration unit at the Viikinmäki wastewater treatment plant in Helsinki (Finland). The developed sensors aim at supporting the existing hardware analyzers by providing a reliable back-up system in case of malfunction of the instruments. In the attempt to design easy to implement and interpretable sensors, computationally light linear models have been considered. However, due to the intrinsic nonlinearity of the process, also nonlinear but still computationally affordable models have been considered for comparison. The experimental results demonstrate the potential of the developed soft-sensors and the possibility for an on-line implementation in the plant's control system as alternative monitoring devices.

Wilson's disease treated with penicillamine and lupus erythematosus: related or distinct entities?

Systemic lupus erythematosus (SLE) has been reported to be associated to Wilson's disease, as a complication of treatment with penicillamine. Even though drug-induced lupus erythematosus (DILE) has some features in common with SLE, they are distinct entities. We report the case of a young girl who at the age of five had a diagnosis of Wilson's disease and she started therapy with penicillamine. Eight years after the beginning of therapy, she developed proteinuria, which was considered to be related to penicillamine. Two years later, she developed arthritis, malar rash and laboratory findings suggestive for lupus erythematosus. At the beginning her symptoms, due to the known association between penicillamine and DILE, were thought to be related to this treatment. In this hypothesis, she was referred to the Rheumatology Centre; zinc acetate was substituted for penicillamine and she started naproxen for the treatment of arthritis. Anyway, the subsequent clinical course and laboratory findings led us to a diagnosis of idiopathic SLE. A renal biopsy detected massive mesangiocapillary proliferation with subendothelial deposits (wire loops) and duplication of glomerular basement membrane (active diffuse global proliferative lupus nephritis, class IV G A). To our knowledge, this is the first report of an association between Wilson's disease and SLE.

Systemic lupus erythematosus presenting with pulmonary thromboembolism in a 15-year-old girl.

Pulmonary embolism is rare in children aged less than 15 years in whom its estimated annual incidence is 0.2 cases per 100,000, against the approximately 700 cases per 100,000 in patients aged more than 85 years. We describe the case of a 15-year-old girl who had a history of thrombocytopenia with anti-platelet and anti-nuclear antibodies. Misdiagnosed chest pain and dyspnea were treated for 6 weeks as community-acquired pneumonia, but proved to be the onset of pulmonary thromboembolism. Although this life-threatening event is rare in paediatrics, the presence of lupus anticoagulant combined with previously asymptomatic thrombocytopenia allowed a diagnosis of systemic lupus erythematosus (SLE). The combination of a history of thrombocytopenia and the presence of antinuclear antibodies, even in the absence of any other diagnostic criteria, should always suggest considering SLE as a differential diagnosis, particularly when the course of a clinical manifestation is atypical.

[Totally laparoscopic total gastrectomy with esophago-jejunal termino-lateral anastomosis by Or-Vil device for carcinoma. Our experience in ten consecutive patients]. / Gastrectomia totale totalmente laparoscopica con anastomosi esofago-digiunale termino-laterale (sistema Or-Vil) nel trattamento del cancro gastrico: nostra esperienza su 10 casi consecutivi.

INTRODUCTION: Laparoscopic gastrectomy represents an alternative procedure for treatment of gastric cancer. Yet, some debate remains about its efficacy, basically from an oncologic point of view. Aim of this study is to analyze our experience with totally laparoscopic total gastrectomy with termino-lateral esophago-jejunal anastomosis by Or-Vil device. PATIENTS AND METHODS: From February 2007 to February 2008, 10 patients underwent the procedure with Or-Vil device for the esophago-jejunal termino-lateral anastomosis. ASA score, UICC-AJCC stage, number of lymph nodes harvested, mortality and morbidity were analyzed. RESULTS: All procedures were concluded laparoscopically. No mortality was observed, morbidity was 20%. Median of lymph nodes harvested was 30+/-14. No port implantations were observed. Discussion. Laparoscopic gastrectomy, both partial and total, is a new challenge, with clear advantages for the patients, but it still must demonstrate its efficacy, especially from the oncologic point of view. In our experience, we can state that totally laparoscopic total gastrectomy is safe, effective and oncologically correct; yet, it is technically demanding and more studies are required to confirm its oncologic efficacy when compared with laparotomic gastrectomy. CONCLUSIONS: Totally laparoscopic total gastrectomy with esophago-jejunal termino-lateral anastomosis by Or-Vil device represents, in our experience, a valid alternative to open procedure; yet, more prospective randomized trials are needed to define this procedure as a new standard for gastric cancer treatment.

Clinical characteristics and cardiac outcome of acute rheumatic fever in Italy in the last 15 years.

OBJECTIVE: To analyze clinical characteristics, cardiac outcome and response to therapy of an Italian pediatric population affected with acute rheumatic fever (ARF) in the last 15 years. METHODS: 135 patients with ARF (aged 2-14.9 years, mean 8.4+/-2.5 years) diagnosed from 1992 to 2006 at the Pediatric Rheumatology Centre of the University of Milan (Italy) were retrospectively reviewed. All patients underwent physical examination, laboratory evaluation, electrocardiogram and echocardiography. Patients were divided into 2 groups: group 1 - patients with complete adherence to updated Jones criteria (107 patients), and group 2 - exceptions (28 patients). Echocardiographic criteria were used to confirm the presence of carditis and to evaluate severity of mitral (MR) and aortic regurgitation (AR) at diagnosis and after treatment with steroids or acetylsalicylic acid (ASA)/non-steroidal anti-inflammatory drugs (NSAIDs). RESULTS: We observed a persistence of ARF in the last 15 years (mean 9 new cases/year with a peak of 19 cases in 2000). Carditis and arthritis were the main major criteria observed (102/135 and 71/135 patients respectively), then chorea (29/135), erythema marginatum (8/135) and subcutaneous nodules (1/135). Arthritis and chorea resolved completely with various therapies. At the last follow-up (> or =5 years) in group 1, loss of MR was observed in 46% steroid-treated (26/56 cases) and in 39% ASA/NSAID-treated (7/18 cases) patients and loss of AR in 59% steroid-treated (22/38) and 2/7 ASA/NSAID-treated patients (p>0.05). CONCLUSION: Incidence of ARF is clinically important currently in the area of northern Italy. Non-suppurative complications of streptococcal pharyngitis should be considered when deciding therapy in a pediatric patient that presents with sore throat.