DNA microarray analysis of Staphylococcus aureus causing bloodstream infection: bacterial genes associated with mortality?

Providing evidence for microbial genetic determinants' impact on outcome in Staphylococcus aureus bloodstream infections (SABSI) is challenging due to the complex and dynamic microbe-host interaction. Our recent population-based prospective study reported an association between the S. aureus clonal complex (CC) 30 genotype and mortality in SABSI patients. This follow-up investigation aimed to examine the genetic profiles of the SABSI isolates and test the hypothesis that specific genetic characteristics in S. aureus are associated with mortality. SABSI isolates (n = 305) and S. aureus CC30 isolates from asymptomatic nasal carriers (n = 38) were characterised by DNA microarray analysis and spa typing. Fisher's exact test, least absolute shrinkage and selection operator (LASSO) and elastic net regressions were performed to discern within four groups defined by patient outcome and characteristics. No specific S. aureus genetic determinants were found to be associated with mortality in SABSI patients. By applying LASSO and elastic net regressions, we found evidence suggesting that agrIII and cna were positively and setC (=selX) and seh were negatively associated with S. aureus CC30 versus non-CC30 isolates. The genes chp and sak, encoding immune evasion molecules, were found in higher frequencies in CC30 SABSI isolates compared to CC30 carrier isolates, indicating a higher virulence potential. In conclusion, no specific S. aureus genes were found to be associated with mortality by DNA microarray analysis and state-of-the-art statistical analyses. The next natural step is to test the hypothesis in larger samples with higher resolution methods, like whole genome sequencing.

Gold and Hydroxyapatite Nano-Composite Scaffolds for Anterior Cruciate Ligament Reconstruction: In Vitro Characterization.

Current anterior cruciate ligament (ACL) graft replacement materials often fail due to the lack of biological integration. While many newly developed extracellular matrix based scaffolds show good biocompatibility they often do not entice cellular remodeling and the rebuilding of a functional ligament. We have proposed the conjugation of gold nanoparticles (AuNP) and hydroxyapatite nanoparticles (nano-HAp) to acellular tissue to enhance cell attachment and proliferation while maintaining an improved degradation resistance and open microstructure. We are the first to investigate the double conjugation of AuNP and nano-HAp onto decellularized tissue to improve the tissue remodeling response. Decellularized porcine diaphragm was crosslinked with two types of nano-HAp and amine-functionalized AuNP with 1-ethyl-3-(3-dimethlaminopropyl) carbodiimide (EDC) crosslinker. Scaffolds were characterized using electron microscopy, differential scanning calorimetry, and fibroblast assays. Results demonstrated that scaffolds with nano-HAp have increased thermal stability at low levels of crosslinking. The open microstructure of the scaffold was not compromised allowing for cell migration while still providing increased degradation resistance. The addition of < 200 nm nano-HAp decreased cell viability compared to scaffolds without nanoparticles, but the addition of AuNP to scaffolds showed enhanced cell viability in the presence of < 200 nm nano-HAp. The addition of < 40 nm nano-HAp showed an increase in cell viability compared to scaffolds crosslinked without nanoparticles. It is concluded that attaching AuNP and < 40nm nano-HAp to extracellular matrices may improve overall properties.

Evaluating the effect of ration composition on income over feed cost and milk yield.

Feed is generally the greatest expense for milk production. With volatility in feed and milk markets, income over feed cost (IOFC) is a more advantageous measure of profit than simply feed cost per cow. The objective of this study was to evaluate the effects of ration cost and ingredient composition on IOFC and milk yield. The Pennsylvania State Extension Dairy Team IOFC tool (http://extension.psu.edu/animals/dairy/business-management/financial-tools/income-over-feed-cost/introduction-to-iofc) was used to collect data from 95 Pennsylvania lactating dairy cow herds from 2009 to 2012 and to determine the IOFC per cow per day. The data collected included average milk yield, milk income, purchased feed cost, ration ingredients, ingredient cost per ton, and amount of each ingredient fed. Feed costs for home-raised feeds for each ration were based on market values rather than on-farm cost. Actual costs were used for purchased feed for each ration. Mean lactating herd size was 170 ± 10.5 and daily milk yield per cow was 31.7 ± 0.19 kg. The mean IOFC was $7.71 ± $1.01 cost per cow, ranging from -$0.33 in March 2009 to $16.60 in September 2011. Data were analyzed using a one-way ANOVA in SPSS (IBM Corp., Armonk, NY). Values were grouped by quartiles and analyzed with all years combined as well as by individual year. Purchased feed cost per cow per day averaged $3.16 ± $1.07 for 2009 to 2012. For 2009 to 2012 combined, milk yield and IOFC did not differ with purchased feed cost. Intermediate levels (quartiles 2 and 3) of forage cost per cow per day between $1.45 and $1.97 per cow per day resulted in the greatest average IOFC of $8.19 and the greatest average milk yield of 32.3 kg. Total feed costs in the fourth quartile ($6.27 or more per cow per day) resulted in the highest IOFC. Thus, minimizing feed cost per cow per day did not maximize IOFC. In 2010, the IOFC was highest at $8.09 for dairies that fed 1 or more commodity by-products. Results of the study indicated that intermediate levels of forage cost and higher levels of total feed cost per cow per day resulted in both higher milk yield and higher IOFC. This suggests that optimal ration formulation rather than least cost strategies may be key to increasing milk yield and IOFC, and that profit margin may be affected more by quality of the feed rather than the cost.

Executive deficits after SARS-CoV-2 infection: A cross-sectional population study.

Importance: Despite the major implications of executive deficits in day-to-day functioning, few studies have investigated this in post-acute sequelae of SARS-CoV-2 infection using standardized measures that differentiate between aspects of executive function. Objective: Examine whether SARS-CoV-2 infection is associated with deficits in executive functions and if so, investigate the duration of this association. Design Setting and Participants: The present research has a cross-sectional design and uses data from the Norwegian Covid-19 Cohort study. The current cohort (n = 8102) completed the Behavior Rating Inventory of Executive Function- Adult Version (BRIEF-A) electronically between April 2021 and September 2021. During the assessment, 4183 of the included participants had a prior positive polymerase chain reaction test (PCR) for SARS-CoV-2 and 3919 were untested or had a confirmed negative PCR test. Exposure: Laboratory-confirmed SARS-CoV-2 infection. Main outcomes and measures: Executive functions were measured using the BRIEF-A, a self-report questionnaire comprising 75 items within nine theoretically and empirically distinct clinical scales. All participants self-reported on demographical variables and comorbidity. Information on sex and age was derived from the personal identification number, and vaccination status was obtained from the Norwegian Immunization Registry (SYSVAK). Results: Participants with a positive SARS-CoV-2 status reported executive deficits in everyday life above the clinical threshold (T-score ≥65) more often than non-infected controls (383 vs. 225). Specifically, the SARS-CoV-2 positive status group indicated significantly more deficits related to metacognition, with the greatest difference demonstrated for working memory. This difference remained when adjusting for various demographic factors and comorbidities, with significantly greater odds of reporting above the clinical threshold following SARS-CoV-2 infection, as observed on the global executive composite score 6-12 months after infection (OR 1.97; 95% CI 1.51 to 2.55). Conclusions: Our study confirms more perceived executive deficits following SARS-CoV-2 infection compared to non-infected controls, with metacognitive aspects being the most affected. These findings shed light on the potential functional difficulties that individuals may encounter during the post-acute phase of SARS-CoV-2 infection and may guide further development of targeted interventions addressing metacognitive domains of executive functioning.

The murine pallid mutation is a platelet storage pool disease associated with the protein 4.2 (pallidin) gene.

Pallid is one of 12 independent murine mutations with a prolonged bleeding time that are models for human platelet storage pool deficiencies in which several intracellular organelles are abnormal. We have mapped the murine gene for protein 4.2 (Epb4.2) to chromosome 2 where it co-localizes with pallid. Southern blot analyses suggest that pallid is a mutation in the Epb4.2 gene. Northern blot analyses demonstrate a smaller than normal Epb4.2 transcript in affected pallid tissues, such as kidney and skin. This is the first gene defect to be associated with a platelet storage pool deficiency, and may allow the identification of a novel structure or biological pathway that influences granulogenesis.

WHO guidelines for the programmatic management of drug-resistant tuberculosis: 2011 update.

The production of guidelines for the management of drug-resistant tuberculosis (TB) fits the mandate of the World Health Organization (WHO) to support countries in the reinforcement of patient care. WHO commissioned external reviews to summarise evidence on priority questions regarding case-finding, treatment regimens for multidrug-resistant TB (MDR-TB), monitoring the response to MDR-TB treatment, and models of care. A multidisciplinary expert panel used the Grading of Recommendations Assessment, Development and Evaluation (GRADE) approach to develop recommendations. The recommendations support the wider use of rapid drug susceptibility testing for isoniazid and rifampicin or rifampicin alone using molecular techniques. Monitoring by sputum culture is important for early detection of failure during treatment. Regimens lasting ≥ 20 months and containing pyrazinamide, a fluoroquinolone, a second-line injectable drug, ethionamide (or prothionamide), and either cycloserine or p-aminosalicylic acid are recommended. The guidelines promote the early use of antiretroviral agents for TB patients with HIV on second-line drug regimens. Systems that primarily employ ambulatory models of care are recommended over others based mainly on hospitalisation. Scientific and medical associations should promote the recommendations among practitioners and public health decision makers involved in MDR-TB care. Controlled trials are needed to improve the quality of existing evidence, particularly on the optimal composition and duration of MDR-TB treatment regimens.

T-lymphocyte heterogeneity in the rat: separation of functional subpopulations using a monoclonal antibody.

W3/25 antibody is the monoclonal product of a hybrid cell resulting from the fusion of a mouse myeloma cell line with spleen cells from a mouse immunized with rat thymocytes. Pure clones have been derived, and segregants free of parental myeloma chains have been isolated. Previous studies have shown that this antibody recognizes a subpopulation of T cells among rat thoracic duct lymphocytes. In the work reported here, three T-cell functions were assayed after separating rat thoracic duct lymphocytes on the fluorescence-activated cell sorter on the basis of labeling with W3/25 antibody. Two of the functional activities appeared to be completely segregated by this procedure. Thus, helper cell activity for an anti-hapten plaque-forming cell response was confined to the labeled population, whereas the allogeneic suppressive effect produced in a parental vector F1 adoptive transfer was mediated by cells in the unlabeled fraction. The third function, graft-versus-host activity, was almost entirely contained within the labeled subpopulation. It is concluded that the antigenic determinant recognized by the monoclonal antibody W3/25 is a differentiation marker for T-cell functional subpopulations.

Role of fluorescence in situ hybridization in sequencing the tomato genome.

The tomato (Solanum lycopersicum L.) genome is being sequenced by a consortium of laboratories in 10 countries. Seventy-seven percent of the tomato genome (DNA) is located in repeat-rich, gene-poor, pericentric heterochromatin, while 23% of the genome is located in repeat-poor, gene-rich, distal euchromatin. It is estimated that approximately 90% of tomato's nuclear genes can be characterized by limiting the sequencing effort to euchromatin while avoiding the problems involved in sequencing the repetitive DNA in heterochromatin. Sequencing is being performed on tomato nuclear DNA cloned into bacterial artificial chromosome (BAC) vectors. Fluorescence in situ hybridization (FISH) is used to help direct the sequencing effort by cytologically demonstrating the location of selected BACs on tomato chromosomes. While mitotic metaphase chromosomes are too short and compact for this purpose, long pachytene chromosomes are ideal. BACs localized in euchromatin can be used confidently as anchors for the assembly of BAC contigs that extend through the euchromatic length of each chromosome arm. Another important role for FISH is identification of BACs near telomeres and near borders with pericentric heterochromatin to indicate that sequencing should not extend much further. This role of FISH is enhanced by our ability to estimate base pair distances between localized BACs and these chromosomal features. Finally, it is noteworthy that when BAC-FISH is combined with chromosomal in situ suppression (CISS) hybridization to block repeats and localize single/low copy sequences, the great majority of BACs localize to single sites. This observation is consistent with tomato being an ancient diploid.

Purkinje cell degeneration associated with erythroid ankyrin deficiency in nb/nb mice.

Mice homozygous for the nb mutation (Chromosome 8) have a severe hemolytic anemia and develop a psychomotor disorder at 6 mo of age. The nb/nb mice are deficient in erythroid ankyrin (Ank-1) but, until the present study, the role of Ank-1 and of Ank-2 (brain ankyrin) in disease genesis was unknown. In normal erythroid tissues, we show that two major transcripts are expressed from Ank-1, and one of these is also present at high levels in the cerebellum. By in situ hybridization and immunocytochemistry, Ank-1 localizes to the cerebellar Purkinje cells and, to a lesser extent, the granule cells. In nb/nb mice, Ank-1 transcripts are markedly reduced in both erythroid and neural tissue, and nb/nb Purkinje cells and granule cells are nearly devoid of Ank-1. The neurological syndrome appears concurrently with a dramatic loss of Purkinje cells. Ank-2 maps to Chromosome 3 and its expression is unaffected by the nb mutation. We conclude that Ank-1 is specifically required for Purkinje cell stability and, in its absence, Purkinje cell loss and neurological symptoms appear.

Replamineform: a new process for preparing porous ceramic, metal, and polymer prosthetic materials.

The replamineform process (meaning replicated life forms) is a technique for duplicating the microstructure of carbonate skeletal components in ceramic, metal, or polymer materials. The special pore structures of marine invertebrate skeletal materials such as echinoid spines and corals, which are difficult or impossible to create artificially, can thus be copied in useful materials. Of immediate interest is the possibility of using these replicated microstructures in the fabrication of orthopedic prosthetic devices. By means of this technique, prosthetic materials having a controlled pore microstructure for optimum strength and tissue ingrowth may be obtained.

Quiet Zone Within a Seismic Gap near Western Nicaragua: Possible Location of a Future Large Earthquake.

A 5700-square-kilometer quiet zone occurs in the midst of the locations of more than 4000 earthquakes off the Pacific coast of Nicaragua. The region is indicated by the seismic gap technique to be a likely location for an earthquake of magnitude larger than 7. The quiet zone has existed since at least 1950; the last large earthquake originating from this area occurred in 1898 and was of magnitude 7.5. A rough estimate indicates that the magnitude of an earthquake rupturing the entire quiet zone could be as large as that of the 1898 event. It is not yet possible to forecast a time frame for the occurrence of such an earthquake in the quiet zone.

Identifying exceptional malaria occurrences in the absence of historical data in South Sudan: a method validation.

BACKGROUND: Detecting unusual malaria events that may require an operational intervention is challenging, especially in endemic contexts with continuous transmission such as South Sudan. Médecins Sans Frontières (MSF) utilises the classic average plus standard deviation (AV+SD) method for malaria surveillance. This and other available approaches, however, rely on antecedent data, which are often missing. OBJECTIVE: To investigate whether a method using linear regression (LR) over only 8 weeks of retrospective data could be an alternative to AV+SD. DESIGN: In the absence of complete historical malaria data from South Sudan, data from weekly influenza reports from 19 Norwegian counties (2006-2015) were used as a testing data set to compare the performance of the LR and the AV+SD methods. The moving epidemic method was used as the gold standard. Subsequently, the LR method was applied in a case study on malaria occurrence in MSF facilities in South Sudan (2010-2016) to identify malaria events that required a MSF response. RESULTS: For the Norwegian influenza data, LR and AV+SD methods did not perform differently (P > 0.05). For the South Sudanese malaria data, the LR method identified historical periods when an operational response was mounted. CONCLUSION: The LR method seems a plausible alternative to the AV+SD method in situations where retrospective data are missing.

European all-cause excess and influenza-attributable mortality in the 2017/18 season: should the burden of influenza B be reconsidered?

OBJECTIVES: Weekly monitoring of European all-cause excess mortality, the EuroMOMO network, observed high excess mortality during the influenza B/Yamagata dominated 2017/18 winter season, especially among elderly. We describe all-cause excess and influenza-attributable mortality during the season 2017/18 in Europe. METHODS: Based on weekly reporting of mortality from 24 European countries or sub-national regions, representing 60% of the European population excluding the Russian and Turkish parts of Europe, we estimated age stratified all-cause excess morality using the EuroMOMO model. In addition, age stratified all-cause influenza-attributable mortality was estimated using the FluMOMO algorithm, incorporating influenza activity based on clinical and virological surveillance data, and adjusting for extreme temperatures. RESULTS: Excess mortality was mainly attributable to influenza activity from December 2017 to April 2018, but also due to exceptionally low temperatures in February-March 2018. The pattern and extent of mortality excess was similar to the previous A(H3N2) dominated seasons, 2014/15 and 2016/17. The 2017/18 overall all-cause influenza-attributable mortality was estimated to be 25.4 (95%CI 25.0-25.8) per 100,000 population; 118.2 (116.4-119.9) for persons aged 65. Extending to the European population this translates into over-all 152,000 deaths. CONCLUSIONS: The high mortality among elderly was unexpected in an influenza B dominated season, which commonly are considered to cause mild illness, mainly among children. Even though A(H3N2) also circulated in the 2017/18 season and may have contributed to the excess mortality among the elderly, the common perception of influenza B only having a modest impact on excess mortality in the older population may need to be reconsidered.

Connectin mediates adhesion in Drosophila.

The Drosophila cell-surface molecule connectin mediates cell-cell adhesion in vitro, and its expression pattern in vivo fits well with an adhesion role in the embryonic neuromuscular system. However, connectin mutants do not show dramatic neuromuscular defects, and ectopic expression studies so far have not supported an adhesion role. Here, we demonstrate that connectin mutants do have a phenotype; the normally connectin-positive pleural muscles fail to adhere closely together. An in vivo adhesion role is supported by misexpression studies, which result in excessive adhesion of normally connectin-negative muscles. Misexpression also causes defects in axon pathfinding. While a previous study interpreted similar defects as indicating a repulsion role for connectin, we argue that the phenotypes are consistent with connectin's adhesion role.

Treatment of idiopathic chylothorax in 14 dogs by ligation of the thoracic duct and partial pericardiectomy.

The outcome and complications associated with thoracic duct ligation combined with partial pericardiectomy in 14 dogs with idiopathic chylothorax were investigated retrospectively. Nine of the dogs were treated in the uk and five in Italy. All of them were reassessed clinically four weeks after surgery and the respiratory function and any pleural fluid accumulation were evaluated; they were followed up by telephone contact for at least six months. Eleven of the dogs were clinically normal and had no radiographic signs of pleural effusion when reassessed after four weeks. Two showed radiographic signs of a minor accumulation of pleural fluid but were clinically normal; when reassessed after three months they showed similar radiographic signs and clinical findings; but after four months there was no evidence of pleural effusion. One dog had a major complication that required a second surgical intervention.

Hypobaric intrathecal anaesthesia for partial hemipelvectomy in a dog.

OBJECTIVE: To report the intrathecal use of a hypobaric anaesthetic solution for partial hemipelvectomy in a nine-year-old, neutered female, Golden Retriever dog, weighing 34 kg. METHODS: Under inhalational anaesthesia, with the dog lying in lateral recumbency and the surgical side uppermost, 1.9 ml of a hypobaric solution containing 3.42 mg of bupivacaine and 0.66 mg of morphine were administered in the subarachnoid space at L5-6 level 30 minutes before surgery. Following the intrathecal injection the dog was maintained for five minutes in a 10 degrees head-down position, then for three minutes in a 10 degrees head-up position. RESULTS: Apart from a transient increase in heart and respiratory rates during resection of the sartorius muscle, which was treated with a plasma Target Controlled Infusion (TCI) of fentanyl, spinal anaesthesia provided cardiovascular stability and excellent relaxation of the surgical site. Neither motor blockade nor proprioceptive deficit were apparent in the contra-lateral hind limb at recovery, 200 minutes after injection. Postoperatively, rescue analgesia was not required in the 48 hours following surgery. CLINICAL SIGNIFICANCE: In dogs, the use of intrathecal hypobaric bupivacaine and morphine as a part of a balanced anaesthetic protocol should be considered during unilateral major orthopaedic surgeries of the pelvis and hind limb, as it allowed a reduction in the dose administered compared to isobaric solutions, providing selective spinal anaesthesia, excellent long-lasting analgesia, and rapid recovery of ambulation.

A novel 16-kilodalton cellular protein physically interacts with and antagonizes the functional activity of c-myc promoter-binding protein 1.

We initially identified c-myc promoter-binding protein 1 (MBP-1) from a human cervical carcinoma cell expression library which negatively regulates c-myc promoter activity. A recent study demonstrated that MBP-1 acts as a general transcriptional repressor (A. K. Ghosh, R. Steele, and R. B. Ray, Mol. Cell. Biol. 19:2880-2886, 1999). In order to identify the cellular protein(s) interacting with MBP-1 for transcriptional regulation, a HeLa cell cDNA expression library was screened using a yeast two-hybrid system. An MBP-1-interacting cDNA encoding a polypeptide of 140 amino acid residues with an approximate molecular mass of 16 kDa was identified and named MBP-1 interacting protein-2A (MIP-2A). MIP-2A has a sequence similarity with an unknown mRNA and SEDL. Mutations in the SEDL gene, located at human chromosome Xp22, has recently been implicated with an X-linked genetic disease, although the function of SEDL gene product was not determined (A. K. Gedeon et al., Nat. Genet. 22:400-404, 1999). However, our results suggested the localization of MIP-2A at human chromosome 19. The specificity of interaction between MBP-1 and MIP-2A was verified by an in vitro glutathione S-transferase pulldown experiment, a mammalian two-hybrid analysis, and in vivo coimmunoprecipitation assays. Further analysis revealed that the amino-terminal domain of MBP-1 (amino acids 1 to 95) interacts with MIP-2A. Immunofluorescent staining suggested colocalization of MIP-2A and MBP-1 primarily in the perinuclear membrane of cells. Functional analysis demonstrated that MIP-2A relieves MBP-1 mediated transcriptional repression on c-myc promoter. Additionally, MIP-2A antagonizes cell growth regulatory role of MBP-1. Taken together, these results suggest the functional interaction of MIP-2A and MBP-1 in cell growth regulation.

Characterization of Mayven, a novel actin-binding protein predominantly expressed in brain.

The cytoskeleton plays an important role in neuronal morphogenesis. We have identified and characterized a novel actin-binding protein, termed Mayven, predominantly expressed in brain. Mayven contains a BTB (broad complex, tramtrack, bric-a-brac)/POZ (poxvirus, zinc finger) domain-like structure in the predicted N terminus and "kelch repeats" in the predicted C-terminal domain. Mayven shares 63% identity (77% similarity) with the Drosophila ring canal ("kelch") protein. Somatic cell-hybrid analysis indicated that the human Mayven gene is located on chromosome 4q21.2, whereas the murine homolog gene is located on chromosome 8. The BTB/POZ domain of Mayven can self-dimerize in vitro, which might be important for its interaction with other BTB/POZ-containing proteins. Confocal microscopic studies of endogenous Mayven protein revealed a highly dynamic localization pattern of the protein. In U373-MG astrocytoma/glioblastoma cells, Mayven colocalized with actin filaments in stress fibers and in patchy cortical actin-rich regions of the cell margins. In primary rat hippocampal neurons, Mayven is highly expressed in the cell body and in neurite processes. Binding assays and far Western blotting analysis demonstrated association of Mayven with actin. This association is mediated through the "kelch repeats" within the C terminus of Mayven. Depolarization of primary hippocampal neurons with KCl enhanced the association of Mayven with actin. This increased association resulted in dynamic changes in Mayven distribution from uniform to punctate localization along neuronal processes. These results suggest that Mayven functions as an actin-binding protein that may be translocated along axonal processes and might be involved in the dynamic organization of the actin cytoskeleton in brain cells.

Gene expression in the developing mouse retina by EST sequencing and microarray analysis.

Retinal development occurs in mice between embryonic day E11.5 and post-natal day P8 as uncommitted neuroblasts assume retinal cell fates. The genetic pathways regulating retinal development are being identified but little is understood about the global networks that link these pathways together or the complexity of the expressed gene set required to form the retina. At E14.5, the retina contains mostly uncommitted neuroblasts and newly differentiated neurons. Here we report a sequence analysis of an E14.5 retinal cDNA library. To date, we have archived 15 268 ESTs and have annotated 9035, which represent 5288 genes. The fraction of singly occurring ESTs as a function of total EST accrual suggests that the total number of expressed genes in the library could approach 27 000. The 9035 ESTs were categorized by their known or putative functions. Representation of the genes involved in eye development was significantly higher in the retinal clone set compared with the NIA mouse 15K cDNA clone set. Screening with a microarray containing 864 cDNA clones using wild-type and brn-3b (-/-) retinal cDNA probes revealed a potential regulatory linkage between the transcription factor Brn-3b and expression of GAP-43, a protein associated with axon growth. The retinal EST database will be a valuable platform for gene expression profiling and a new source for gene discovery.