Evaluation of SDS and GRAS liquid disinfectants for mitigation of hepatitis A virus contamination of berries.

AIM: To evaluate generally recognized as safe (GRAS) liquid wash formulations against hepatitis A virus-contaminated strawberries and blackberries in order to identify a formulation suitable for reducing virus contamination. METHODS AND RESULTS: Formulations included the surfactant sodium dodecyl sulfate (SDS; 0·5% w/v) by itself, and in combination, with lactic acid (LA; 0·5% v/v), levulinic acid (LVA; 0·5% v/v) and 3 ppm aqueous chlorine dioxide (ClO2 ). After contamination and drying overnight, the average total extracted contamination for both untreated strawberries and blackberries was 4·4 log PFU. Three successive distilled H2 O only treatments reduced total contamination by up to 1·8 log PFU for both strawberries and blackberries, while wash formulations showed significant (P ≤ 0·05) total reductions ranging from 2·1 to 2·9 log PFU. CONCLUSIONS: Considering results for both berry types, the combination of ClO2 and SDS was the most effective. Overall results indicate that adding surfactant and several types of sanitizers to berry wash can enhance HAV reduction on berries. SIGNIFICANCE AND IMPACT OF THE STUDY: This study indicates that industry could enhance the virologic safety of ready-to-eat berries by the combined use of surfactant and sanitizer.

Evaluation of 405-nm monochromatic light for inactivation of Tulane virus on blueberry surfaces.

AIM: The study aim was to evaluate the potential of 405-nm light as a virus intervention for blueberries. METHODS AND RESULTS: Tulane virus (TV)-inoculated blueberries were treated with 4·2 mW cm-2 of 405-nm light for 5-30 min. To mitigate thermal heating due to the intense light, a dry ice-chilled, nitrogen-based cooling system was utilized. Blueberries were rotated to ensure exposure of all surfaces to 405-nm light. Five-, 15- and 30-min treatments resulted in little or no inactivation of TV on blueberries (average log reductions of -0·18; -0·02; and +0·06 respectively). Since 405-nm light's inactivation mechanism may involve singlet oxygen, two singlet oxygen enhancers, riboflavin and rose bengal, were used to coat the blueberries prior to 405-nm light treatment. When 0·1% riboflavin or rose bengal was added, resulting in an average PFU reduction of -0·51 and -1·01 logs respectively. However, it was noted that the addition of riboflavin and rose bengal in the absence of 405-nm light treatment produced some inactivation. Average untreated log reductions for riboflavin and rose bengal were -0·13 and -0·66 respectively. Also, 60-30-s 405-nm light pulses with 2-min ambient cooling periods without the dry ice nitrogen cooling system did not inactivate TV, suggesting that oxygen limitation by the nitrogen CO2 mixture was not the cause of limited inactivation. CONCLUSIONS: Overall results indicate that 405-nm light has some potential to inactivate viruses if singlet oxygen enhancers are present. SIGNIFICANCE AND IMPACT OF THE STUDY: The potential of visible monochromatic violet/blue light (405 nm) as a nonthermal intervention for viruses on foods, such as berries that are prone to norovirus contamination, had not been previously evaluated. Use of food-grade singlet oxygen enhancer compounds in combination with visible spectra light may offer a means to inactivate foodborne viruses.

Outbreak of Hepatitis A in Italy Associated with Frozen Redcurrants Imported from Poland: A Case Study.

Hepatitis A virus (HAV) was detected in a batch of imported non-packaged frozen redcurrants purchased in a Bari grocery. Sequence and phylogenetic analysis showed the HAV strain clustered tightly with the HAV strain from the 2013 Italian epidemic, providing additional evidence that frozen redcurrants were the main vehicle of the HAV outbreak.

Single-step laser-based fabrication and patterning of cell-encapsulated alginate microbeads.

Alginate can be used to encapsulate mammalian cells and for the slow release of small molecules. Packaging alginate as microbead structures allows customizable delivery for tissue engineering, drug release, or contrast agents for imaging. However, state-of-the-art microbead fabrication has a limited range in achievable bead sizes, and poor control over bead placement, which may be desired to localize cellular signaling or delivery. Herein, we present a novel, laser-based method for single-step fabrication and precise planar placement of alginate microbeads. Our results show that bead size is controllable within 8%, and fabricated microbeads can remain immobilized within 2% of their target placement. Demonstration of this technique using human breast cancer cells shows that cells encapsulated within these microbeads survive at a rate of 89.6%, decreasing to 84.3% after five days in culture. Infusing rhodamine dye into microbeads prior to fluorescent microscopy shows their 3D spheroidal geometry and the ability to sequester small molecules. Microbead fabrication and patterning is compatible with conventional cellular transfer and patterning by laser direct-write, allowing location-based cellular studies. While this method can also be used to fabricate microbeads en masse for collection, the greatest value to tissue engineering and drug delivery studies and applications lies in the pattern registry of printed microbeads.

The progressive ankylosis protein regulates cementum apposition and extracellular matrix composition.

BACKGROUND/AIMS: Tooth root cementum is sensitive to modulation of inorganic pyrophosphate (PP(i)), an inhibitor of hydroxyapatite precipitation. Factors increasing PP(i) include progressive ankylosis protein (ANK) and ectonucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) while tissue nonspecific alkaline phosphatase hydrolyzes PP(i). Studies here aimed to define the role of ANK in root and cementum by analyzing tooth development in Ank knock-out (KO) mice versus wild type. MATERIALS AND METHODS: Periodontal development in KO versus control mice was analyzed by histology, histomorphometry, immunohistochemistry, in situ hybridization, electron microscopy, and nanoindentation. Cementoblast cultures were used in vitro to provide mechanistic underpinnings for PP(i) modulation of cell function. RESULTS: Over the course of root development, Ank KO cervical cementum became 8- to 12-fold thicker than control cervical cementum. Periodontal ligament width was maintained and other dentoalveolar tissues, including apical cementum, were unaltered. Cervical cementum uncharacteristically included numerous cells, from rapid cementogenesis. Ank KO increased osteopontin and dentin matrix protein 1 gene and protein expression, and markedly increased NPP1 protein expression in cementoblasts but not in other cell types. Conditional ablation of Ank in joints and periodontia confirmed a local role for ANK in cementogenesis. In vitro studies employing cementoblasts indicated that Ank and Enpp1 mRNA levels increased in step with mineral nodule formation, supporting a role for these factors in regulation of cementum matrix mineralization. CONCLUSION: ANK, by modulating local PP(i), controls cervical cementum apposition and extracellular matrix. Loss of ANK created a local environment conducive to rapid cementogenesis; therefore, approaches modulating PP(i) in periodontal tissues have potential to promote cementum regeneration.

The genetic basis of divergent pigment patterns in juvenile threespine sticklebacks.

Animal pigment patterns are important for a range of functions, including camouflage and communication. Repeating pigment patterns, such as stripes, bars and spots have been of particular interest to developmental and theoretical biologists, but the genetic basis of natural variation in such patterns is largely unexplored. In this study, we identify a difference in a periodic pigment pattern among juvenile threespine sticklebacks (Gasterosteus aculeatus) from different environments. Freshwater sticklebacks exhibit prominent vertical bars that visually break up the body shape, but sticklebacks from marine populations do not. We hypothesize that these distinct pigment patterns are tuned to provide crypsis in different habitats. This phenotypic difference is widespread and appears in most of the freshwater populations that we sampled. We used quantitative trait locus (QTL) mapping in freshwater-marine F2 hybrids to elucidate the genetic architecture underlying divergence in this pigmentation pattern. We identified two QTL that were significantly associated with variation in barring. Interestingly, these QTL were associated with two distinct aspects of the pigment pattern: melanophore number and overall pigment level. We compared the QTL locations with positions of known pigment candidate genes in the stickleback genome. We also identified two major QTL for juvenile body size, providing new insights into the genetic basis of juvenile growth rates in natural populations. In summary, although there is a growing literature describing simple genetic bases for adaptive coloration differences, this study emphasizes that pigment patterns can also possess a more complex genetic architecture.

High hydrostatic pressure processing of murine norovirus 1-contaminated oysters inhibits oral infection in STAT-1(-/-)-deficient female mice.

We have previously demonstrated that high pressure processing (HPP) is effective in preventing in vitro replication of murine norovirus strain 1 (MNV-1), a human norovirus surrogate, in a monocyte cell line following extraction from MNV-1-contaminated oysters. In the present study, the efficacy of HPP to prevent in vivo replication within mice fed HPP-treated MNV-1-seeded oyster extracts was evaluated. Oyster homogenate extracts seeded with MNV-1 were given 5-min, 400-MPa (58,016-psi) treatments and orally gavaged into immunodeficient (STAT-1(-/-)) female mice. Mice orally gavaged with HPP-treated MNV-1 showed significant (P ≤ 0.05) weight loss leading to enhanced morbidity, whereas those given 100 and 200 PFU of HPP-treated MNV-1 were comparable to uninfected controls. MNV-1 was detected, via real-time PCR, within the liver, spleen, and brain of all mice fed non-HPP-treated homogenate but was not detected in the tissues of mice fed HPP-treated homogenates or in uninfected control mice. Hepatocellular necrosis and lymphoid depletion in the spleen were observed in non-HPP-treated MNV-1 mice only. These results clearly show that HPP prevents MNV-1 infection in vivo and validates that viral inactivation by HPP in vitro is essentially equivalent to that in vivo. Further, the data suggest that HPP may be an effective food processing intervention for norovirus-contaminated shellfish and thus may decrease risk to both immunocompromised and immunocompetent individuals who consume shellfish.

Cell yields and fermentation responses of a Salmonella Typhimurium poultry isolate at different dilution rates in an anaerobic steady state continuous culture.

The objectives of these studies were to determine cell yield and fermentation responses of a Salmonella enterica serovar Typhimurium poultry isolate using various dilution rates in steady state continuous culture incubations. S. enterica Typhimurium cells were propagated in continuous cultures with a total volume of 0.50 l of Luria Bertani medium containing 0.1% glucose. Dilution rates from 0.0125 to 1.44/h were used. Cell protein concentration generally increased linearly with increased dilution rate up to a rate of 0.54/h and declined at the higher dilution rates. Glucose consumption gave a similar pattern to cell protein concentration by declining at the three highest dilution rates. Short chain fatty acid production was inconsistently influenced by dilution rate. Acetate, the most predominant fatty acid produced, declined at the higher dilution rates, as did propionate. Ammonia production remained stable at the lowest dilution rates, but increased significantly at a dilution rates above 0.27/h.

Retrograde amnesia and the volume of critical brain structures.

There are many controversies concerning the structural basis of retrograde amnesia (RA). One view is that memories are held briefly within a medial temporal store ("hippocampal complex") before being "consolidated" or reorganised within temporal neocortex and/or networks more widely distributed within the cerebral cortex. An alternative view is that the medial temporal lobes are always involved in the storage and retrieval (reactivation) of autobiographical memories (multiple trace theory). The present study used quantitative magnetic resonance imaging (MRI) in 40 patients with focal pathology/volume loss in different sites, to examine the correlates of impairment on three different measures of RA. The findings supported the view that widespread neural networks are involved in the storage and retrieval of autobiographical and other remote memories. Brain volume measures in critical structures could account for 60% of variance on autobiographical memory measures (for incidents and facts) in diencephalic patients and for 60-68% of variance in patients with frontal lesions. Significant correlations with medial temporal lobe volume were found only in the diencephalic group, in whom they were thought to reflect thalamic changes, but not in patients with herpes encephalitis or hypoxia in whom the temporal lobes were particularly implicated. The latter finding fails to support one of the main predictions of multiple trace theory, as presently expounded.

Cementum: a phosphate-sensitive tissue.

Ectopic calcification within joints has been reported in humans and rodents exhibiting mutations in genes that regulate the level of extracellular pyrophosphate, e.g., ank and PC-1; however, periodontal effects of these mutations have not previously been examined. These initial studies using ank and PC-1 mutant mice were done to see if such mineral deposition and resulting ankylosis were occurring in the periodontium as well. Surprisingly, results indicated the absence of ankylosis; however, a marked increase in cementum formation on the root surfaces of fully developed teeth of these mutant mice was noted. Examination of ank mutant mice at earlier ages of tooth root formation indicated that this striking observation is apparent from the onset of cementogenesis. These findings suggest that cells within the periodontal region are highly responsive to changes in phosphate metabolism. This information may prove valuable in attempts to design successful therapies for regenerating periodontal tissues.

Reciprocal mouse and human limb phenotypes caused by gain- and loss-of-function mutations affecting Lmbr1.

The major locus for dominant preaxial polydactyly in humans has been mapped to 7q36. In mice the dominant Hemimelic extra toes (Hx) and Hammertoe (Hm) mutations map to a homologous chromosomal region and cause similar limb defects. The Lmbr1 gene is entirely within the small critical intervals recently defined for both the mouse and human mutations and is misexpressed at the exact time that the mouse Hx phenotype becomes apparent during limb development. This result suggests that Lmbr1 may underlie preaxial polydactyly in both mice and humans. We have used deletion chromosomes to demonstrate that the dominant mouse and human limb defects arise from gain-of-function mutations and not from haploinsufficiency. Furthermore, we created a loss-of-function mutation in the mouse Lmbr1 gene that causes digit number reduction (oligodactyly) on its own and in trans to a deletion chromosome. The loss of digits that we observed in mice with reduced Lmbr1 activity is in contrast to the gain of digits observed in Hx mice and human polydactyly patients. Our results suggest that the Lmbr1 gene is required for limb formation and that reciprocal changes in levels of Lmbr1 activity can lead to either increases or decreases in the number of digits in the vertebrate limb.

Evaluation of cervical spine in intensive care patients following blunt trauma.

The aim of this study was to review a Level 1 trauma center's use of early (< 72 hours from injury) limited MRI to "clear" cervical spine extradural soft tissue injuries in ICU patients sustaining blunt trauma. A retrospective review of the records of patients meeting entry criteria during 1997 was performed. Demographic data, cervical spine radiographic and imaging evaluation, results, and follow-up information were gathered. One hundred and fifty patients met criteria. Forty-one patients had initial static radiographs that revealed cervical spine trauma. Twenty-seven of the 108 patients with normal initial static radiographs had evidence of extradural soft tissue injury on MRI indicating potential spinal column instability. Twenty-one of the 108 patients had negative MRI and were liberated from cervical spine precautions at a mean of 2.9 +/- 0.9 days from injury. The remaining patients were cleared of cervical spine precautions by plain radiographs and reliable clinical examinations, or by dynamic radiographs, or they died before complete evaluation. The diagnosis of acute injury to the cervical spine from blunt trauma in ICU patients must include evaluation of the osseous spine and extradural soft tissues. Dynamic studies such as flexion and extension views place the obtunded ICU patient at risk of potential neurologic injury. MRI is a noninvasive imaging technique that allows evaluation of extradural soft tissue injury with potentially less patient risk and with fewer personnel. MRI allows early liberation of cervical spine precautions in those patients with negative studies. Further studies are needed to compare specific ligamentous injury patterns by MRI with dynamic studies of the C-spine to further define MRI injury patterns indicating risk of acute spinal instability.

Rapid and efficient extraction method for reverse transcription-PCR detection of hepatitis A and Norwalk-like viruses in shellfish.

As part of an effort to develop a broadly applicable test for Norwalk-like viruses and hepatitis A virus (HAV) in shellfish, a rapid extraction method that is suitable for use with one-step reverse transcription (RT)-PCR-based detection methods was developed. The method involves virus extraction using a pH 9.5 glycine buffer, polyethylene glycol (PEG) precipitation, Tri-reagent, and purification of viral poly(A) RNA by using magnetic poly(dT) beads. This glycine-PEG-Tri-reagent-poly(dT) method can be performed in less than 8 h on hard-shell clams (Mercenaria mercenaria) and Eastern oysters (Crassostrea virginica) and, when coupled with RT-PCR-based detection, can yield results within 24 h. Observed sensitivities for seeded shellfish extracts are as low as 0.015 PFU of HAV and 22.4 RT-PCR50 units for Norwalk virus. Detection of HAV in live oysters experimentally exposed to contaminated seawater is also demonstrated. An adaptation of this method was used to identify HAV in imported clams (tentatively identified as Ruditapes philippinarum) implicated in an outbreak of food-borne viral illness. All of the required reagents are commercially available. This method should facilitate the implementation of RT-PCR testing of commercial shellfish.

Rare complications of endoscopic retrograde cholangiopancreatography: two case reports.

BACKGROUND: Endoscopic retrograde cholangiopancreatography (ERCP) is a diagnostic procedure with several known risks. We present two rarely reported complications of ERCP and sphincterotomy: transverse mesocolon disruption with ischemic colitis and splenic rupture. RESULTS: The first patient, a 54-year-old female, presented one day following ERCP and stent revision for pancreas divisum. She presented with hypotension and abdominal distention. An abdominal computed tomography (CT) showed a ruptured spleen, which was confirmed on laparotomy. She had a complicated postoperative course and died of multiple organ failure. The second patient is a 56-year-old female who presented five days after ERCP and sphincterotomy with abdominal pain, abdominal wall ecchymosis, and decreasing hematocrit. Her evaluation included hospital admission and abdominal CT scan, which showed free fluid and a large hematoma in the transverse mesocolon. These findings were confirmed on laparotomy and a devascularized segment of bowel was resected. CONCLUSION: Only 6 cases of ERCP-related splenic injury have been reported in the literature. One additional report is available of a fatal splenic artery injury. No previous reports exist of a mesenteric hematoma resulting in bowel devascularization. Prompt evaluation and awareness of potential complications should help capture potentially life-threatening sequelae of ERCP.

Structural MRI volumetric analysis in patients with organic amnesia, 2: correlations with anterograde memory and executive tests in 40 patients.

BACKGROUND: Cognitive-MRI correlations have often been studied in disorders in which there are multiple cognitive deficits and widespread cortical atrophy, such as Alzheimer's dementia. In such circumstances, the interpretation of any single cognitive-structural correlation is equivocal. Only by measuring differing cognitive functions and a wide range of brain structures in patients with a varying distribution of lesions or atrophy can specific brain-cognitive relations be determined in neurological disorder. METHOD: In the present study, a clear set of anatomical criteria and detailed MRI segmentation procedures were applied to measure whole brain, and left and right frontal, temporal lobe, anterolateral and medial temporal volumes, as well as thalamic cross sectional areas in 40 patients with organic amnesia (from various diseases) and 10 healthy controls. RESULTS: Within the total patient group, anterograde memory measures correlated significantly with medial temporal, hippocampal, and thalamic measurements. A spatial memory measure correlated significantly with hippocampal volume, and temporal context memory with frontal volume. After a factor analysis of the cognitive measures, the association between anterograde memory and hippocampal volume was corroborated. Forgetting rates and subjective memory evaluations did not show any significant MR correlations and, of executive tests employed, only card sorting categories correlated significantly with frontal volume. CONCLUSION: Loss of volume in key brain structures (for example, hippocampus, thalamus) is detectable on quantitative MRI, and this loss of volume correlates significantly with impaired performance on measures of anterograde memory function. Correlations with hippocampal volume did not indicate a specific role in either recall or verbal memory, as opposed to recognition or visual memory.

Structural MRI volumetric analysis in patients with organic amnesia, 1: methods and comparative findings across diagnostic groups.

BACKGROUND: If they are to be replicable, MRI volume measurements require explicit definitions of structures and of criteria for delineating these structures on MRI. Previously published volumes in healthy subjects show considerable differences in measurements across different studies, including a fourfold variation in estimates of hippocampal volume. Previous neuroimaging reports in patients with Korsakoff syndrome have generally found widespread or non-specific change, whereas in patients with herpes encephalitis the extent of pathological involvement reported beyond the temporal lobes has varied. METHOD: In the present study, a clear set of anatomical criteria and detailed MRI segmentation procedures were applied to measure whole brain, frontal and temporal lobe, and anterolateral and medial temporal volumes, as well as thalamic areas in patients with organic amnesia (from Korsakoff's syndrome, herpes encephalitis, and focal frontal lesions) as well as healthy controls. RESULTS: Patients with Korsakoff's syndrome showed decreased thalamic measurements but no significant changes in the medial temporal lobes, whereas patients with herpes encephalitis showed severe medial temporal but not thalamic atrophy. In the patients with known frontal lobe lesions, quantitative analysis on MRI showed reduced frontal lobe volume but no significant temporal lobe or thalamic atrophy. CONCLUSION: Quantified MRI can be a useful technique with which to examine brain-cognitive relations, provided that detailed techniques are explicitly described. In particular, specific patterns of volume change can be found in vivo in patients with Korsakoff's syndrome and those with herpes encephalitis.

The BMP family member Gdf7 is required for seminal vesicle growth, branching morphogenesis, and cytodifferentiation.

Epithelial-mesenchymal interactions play an important role in the development of many different organs and tissues. The secretory glands of the male reproductive system, including the prostate and seminal vesicles, are derived from epithelial precursors. Signals from the underlying mesenchyme are required for normal growth, branching, and differentiation of the seminal vesicle epithelium. Here, we show that a member of the BMP family, Gdf7, is required for normal seminal vesicle development. Expression and tissue recombination experiments suggest that Gdf7 is a mesenchymal signal that acts in a paracrine fashion to control the differentiation of the seminal vesicle epithelium.

Genetic control of bone and joint formation.

The form and pattern of the vertebrate skeleton is thought to be strongly influenced by several fundamental morphogenetic behaviours of mesenchymal cells during embryonic development. Recent genetic and developmental studies have identified some of the genes that play an important role in controlling both the aggregation of mesenchymal cells into rough outlines of future skeletal elements (condensations), and in controlling where skeletal precursors cleave or segment to produce separate skeletal elements connected by joints. Members of the bone morphogenetic protein (BMP) family appear to play an important role in both processes. Mouse and human mutations in these genes lead to defects in formation of specific bones and joints, with striking specificity for particular anatomical locations. Results from a range of experiments suggest that these molecules may have multiple functions during normal skeletal development and patterning. A major challenge for the future is to identify genes and pathways that can maintain, repair, or stimulate the regeneration of bone and joint structures at later developmental stages.

A large-scale in situ screen provides molecular evidence for the induction of eye anterior segment structures by the developing lens.

The anterior segment of the vertebrate eye includes the cornea, iris, ciliary body, trabecular meshwork, and lens. Although malformations of these structures have been implicated in many human eye diseases, little is known about the molecular mechanisms that control their development. To identify genes involved in anterior segment formation, we developed a large-scale in situ hybridization screen and examined the spatial and temporal expression of over 1000 genes during eye development. This screen identified 62 genes with distinct expression patterns in specific eye structures, including several expressed in novel patterns in the anterior segment. Using these genes as developmental markers, we tested for the presence of inductive signals that control the differentiation of anterior segment tissues. Organ culture recombination experiments showed that a chick lens is capable of inducing the expression of markers of the presumptive iris and ciliary body in the developing mouse neural retina. The inducing activity from the lens acts only over short ranges and is present at multiple stages of eye development. These studies provide molecular evidence that an evolutionarily conserved signal from the lens controls tissue specification in the developing optic cup.