Routine testing of close contacts of confirmed COVID-19 cases - National COVID-19 Contact Management Programme, Ireland, May to August 2020.

OBJECTIVES: The objective of this study was to inform public health practitioners who are designing, adapting and implementing testing and tracing strategies for Coronavirus disease (COVID-19) control. STUDY DESIGN: The study design is monitoring and evaluation of a national public health protection programme. METHODS: All close contacts of laboratory-confirmed cases of COVID-19 identified between the 19th May and 2nd August were included; secondary attack rates and numbers needed to test were estimated. RESULTS: Four thousand five hundred eighty six of 7272 (63%) close contacts of cases were tested with at least one test. The secondary attack rate in close contacts who were tested was 7% (95% Confidence Interval [CI]: 6.3 - 7.8%). At the 'day 0' test, 14.6% (95% CI: 11.6-17.6%) of symptomatic close contacts tested positive compared with 5.2% (95% CI: 4.4-5.9%) of asymptomatic close contacts. CONCLUSIONS: The application of additional symptom-based criteria for testing in this high-incidence population (close contacts) is of limited utility because of the low negative predictive value of absence of symptoms.

Predicting pork loin chop yield using carcass and loin characteristics.

The objective was to determine the predictive ability of carcass length for the number of equal-thickness chops obtained from a boneless pork loin. Longer pork carcasses are assumed to yield longer loins and, therefore, an increased number of chops. Loins were collected from pigs (1,238 total) raised under commercial conditions and marketed when the mean pig weight in a pen reached 138 kg. Pigs were slaughtered over 7 wk in a commercial facility. Carcass length was measured at 1 d postmortem on the left side of each carcass from the anterior edge of the symphysis pubis bone to the anterior edge of the first rib. Carcasses were fabricated, and boneless loins (North American Meat Processors number 414) were vacuum packaged and transported to the University of Illinois Meat Science Laboratory. Loins were stored at 4°C for 14 d. At the end of the aging period, loins were weighed, measured for stretched length (stretched to maximum length without distortion) and compressed length (compressed to minimum length without distortion), and sliced into 2.54-cm-thick chops. Boneless chops were counted and weighed. Carcass length ranged from a minimum of 78.2 cm to a maximum of 96.5 cm and the number of boneless chops ranged from a minimum of 13 to a maximum of 20 chops. Data were analyzed using the regression procedure of SAS. The dependent variable was the number of boneless chops. Coefficient of determination () was calculated for carcass length, boneless loin weight, compressed loin length, and stretched loin length. Carcass length explained 15% ( < 0.0001) of the variation in the number of loin chops. Loin weight explained 33% ( < 0.0001) of the variation in the number of loin chops. Compressed loin length and stretched loin length explained 28 and 8% ( < 0.0001), respectively, of the variation in the number of loin chops. Multiple linear regression was used to determine a predictive equation for the number of loin chops using the stepwise selection option of all independent variables. The combination of boneless loin weight, compressed loin length, 10th-rib carcass fat depth, and carcass length explained 45% of the variation ( < 0.0001; C(p) = 16.76) in the number of loin chops using a required statistic at the SLENTRY and SLSTAY level = 0.15. Overall, carcass length is a poor predictor of the number of equal-thickness loin chops that can be derived from a boneless pork loin.

Optical coherence tomography characteristics of different types of big bubbles seen in deep anterior lamellar keratoplasty by the big bubble technique.

PurposeTo define optical coherence tomography (OCT) characteristics of type-1, type-2, and mixed big bubbles (BB) seen in deep anterior lamellar keratoplasty.MethodsHuman sclero-corneal discs were obtained from UK (30) and Canada (16) eye banks. Air was injected into corneal stroma until a BB formed. UK samples were fixed in formalin before scanning with Fourier-domain (FD-OCT). One pair of each type of BB was scanned fresh. All BB obtained from Canada were scanned fresh with time-domain (TD-OCT). For each OCT machine used, type-1 BB from which Descemets membrane (DM) was partially peeled, were also scanned. The morphological characteristics of the scans were studied.ResultsFD-OCT of the posterior wall of type-1 (Dua's layer (DL) with DM) and type-2 BB (DM alone) both revealed a double-contour hyper-reflective curvilinear image with a hypo-reflective zone in between. The anterior line of type-2 BB was thinner than that seen with type-1 BB. In mixed BB, FD-OCT showed two separate curvilinear images. The anterior image was a single hyper-reflective line (DL), whereas the posterior image, representing the posterior wall of type-2 BB (DM) was made of two hyper-reflective lines with a dark space in between. TD-OCT images were similar with less defined component lines, but the entire extent of the BB could be visualised.ConclusionOn OCT examination the DM and DL present distinct features, which can help identify type-1, type-2, and mixed BB. These characteristics will help corneal surgeons interpret intraoperative OCT during lamellar corneal surgery.

Effects of marketing group on the quality of fresh and cured hams sourced from a commercial processing facility.

The objective was: 1) to characterize the effect of marketing group on fresh and cured ham quality, and 2) to determine which fresh ham traits correlated to cured ham quality traits. Pigs raised in 8 barns representing 2 seasons (hot and cold) and 2 production focuses (lean and quality) were used. Three groups were marketed from each barn. A total of 7,684 carcasses were used for data collection at the abattoir. Every tenth carcass was noted as a select carcass for in-depth ham quality analyses. Leg primal weight and instrumental color were measured on 100% of the population. On the select 10% of the population, hams were fabricated into sub-primal pieces, and 3-piece hams were manufactured to evaluate cured ham quality and processing yield. Data were analyzed as a split-plot design in the MIXED procedure of SAS with production focus as the whole-plot factor, and marketing group as the split-plot factor. Pearson correlation coefficients between fresh and cured ham traits were computed. There were no differences ( ≥ 0.15) in instrumental color or ultimate pH ( ≥ 0.14) among fresh ham muscles from any marketing group. The only exception was the semimembranosus of marketing group 2 was lighter than marketing group 1 ( = 0.03) and the dark portion of the semitendinosus muscle from group 1 was lighter than from group 3 ( = 0.01). There were no differences ( ≥ 0.33) in ultimate pH of fresh ham muscles between production focuses, but several muscles from quality focus pigs were lighter in color than ham muscles from lean focus pigs. The lack of differences in fresh ham quality lead to few differences in cured ham quality. Cured hams from the quality focus pigs had greater lipid content ( < 0.01) than hams from lean focus pigs. Cured lightness values of hams from marketing group 1 and 2 were 1.52 units lighter than hams from marketing group 3 ( 0.01). Overall, marketing group did not impact ham quality. Fresh ham quality was not strongly related to cured ham quality. Some correlations were present between fresh and cured ham traits, but those relationships were likely not strong enough to be used as a sorting tool for fresh hams to generate high quality cured hams.

Pork loin quality is not indicative of fresh belly or fresh and cured ham quality.

The objective was to characterize the relationship between fresh loin quality with fresh belly or fresh and cured ham quality. Pigs raised in 8 barns representing 2 seasons [cold ( = 4,290) and hot ( = 3,394)] and 2 production focuses [lean ( = 3,627) and quality ( = 4,057)] were used. Carcass characteristics and other meat quality data were collected on 7,684 carcasses. All of the carcasses were evaluated for HCW, LM depth, tenth rib fat depth, leg (ham primal) weight, instrumental color on the gluteus medius and gluteus profundus of the ham face, and subjective loin quality. Instrumental loin color and ultimate pH (≥ 22 h postmortem) were collected on the ventral side of loins along with dimensions and firmness scores of fresh bellies from 50% of the carcasses. Ten percent of the boneless loins and fresh hams were evaluated for slice shear force (SSF) or cured ham characteristics. Correlation coefficients between traits were computed using the CORR procedure of SAS and considered significantly different from 0 at ≤ 0.05. Temperature decline, beginning at 31 min postmortem and concluding at 22 h postmortem, for the longissimus dorsi and semimembranosus muscles were evaluated on 10% of the carcasses. Ultimate loin pH was correlated with dimensional belly characteristics ( ≥ |0.07|; < 0.0001) fresh ham instrumental color ( ≥ |0.03|; ≤ 0.05), and semimembranosus ultimate pH ( = 0.33; < 0.0001). Further, ultimate loin pH was correlated ( ≤ 0.01) with pump retention ( = 0.087) and cooked yield ( = 0.156) of cured hams. Instrumental L*on the ventral surface of the loin was related to L* on both muscles of the ham face ( ≤ 0.0001). Even though significant relationships between the loin, belly, and ham were detected, the variability in belly and ham quality explained by variability in loin quality was poor (≤ 22.09%). Compositional differences between the loin and belly may have contributed to those poor relationships. Additionally, differences in temperature declines during chilling between the loin and ham likely contributed to the weak nature of relationships. Equilibration of longissimus dorsi temperature to ambient cooler temperature occurred at 14 h postmortem ( = 0.0005), yet the semimembranosus had not equilibrated with ambient (equilibration bay) temperature ( < 0.0001) at 22 h postmortem. Using loin quality to draw conclusions about fresh belly and fresh and cured ham quality may be misleading.

Chronic treatment with a tryptophan-rich protein hydrolysate improves emotional processing, mental energy levels and reaction time in middle-aged women.

Common pharmacological treatments of mood disorders aim to modulate serotonergic neurotransmission and enhance serotonin levels in the brain. Brain serotonin levels are dependent on the availability of its food-derived precursor essential amino acid tryptophan (Trp). We tested the hypothesis that delivery of Trp via food may serve as an alternative treatment, and examined the effects of a Trp-rich, bioavailable dietary supplement from egg protein hydrolysate on cognitive and emotional functions, mood state, and sleep quality. In a randomised, placebo-controlled, parallel trial, fifty-nine mentally and physically healthy women aged 45-65 years received placebo (n 30) or the supplement (n 29) (both as 0.5 g twice per d) for 19 d. Emotional processing was significantly changed by supplementation, exhibiting a shift in bias away from negative stimuli. The results for the Affective Go/No-Go Task exhibited a slowing of responses to negative words, suggesting reduced attention to negative emotional stimuli. The results for the Facial Emotional Expression Rating Task also supported a shift away from attention to negative emotions and a bias towards happiness. An increase in arousal-like symptoms, labelled 'high energy', shorter reaction times and a slight benefit to sustained attention were observed in the treated subjects. Finally, when the supplement was taken 60-90 min before bedtime, a feeling of happiness before going to bed was consistently reported. In summary, daily consumption of a low-dose supplement containing bioavailable Trp may have beneficial effects on emotional and cognitive functions.

Effects of acute treatment with a tryptophan-rich protein hydrolysate on plasma amino acids, mood and emotional functioning in older women.

RATIONALE: Effective functioning of the neurotransmitter serotonin is important for optimal cognitive and emotional function. Dietary supplements able to increase availability to the brain of the precursor amino acid, tryptophan (TRP), and thereby enhance serotonin synthesis, can have measurable impact on these psychological processes. OBJECTIVES: This study involves a randomised controlled trial of a TRP-rich egg-white protein hydrolysate (DSM Nutritional Products Ltd., Switzerland) on plasma amino acids, cognition, mood and emotional processing in older women. METHODS: Following a baseline test day without treatment, 60 healthy women aged 45-65 years received drinks containing either 2 or 4 g of TRP-rich protein hydrolysate product or 3.11 g casein hydrolysate as a control. One hour later, they undertook a 2-h battery of cognitive and emotional tests. RESULTS: The TRP-rich protein hydrolysate produced the expected dose-dependent increase in the ratio of plasma TRP to competing large neutral amino acids. TRP-rich protein hydrolysate (2 g only) prevented both the decline in wellbeing and increase in fatigue seen over the test session in the control group. This treatment dose resulted in a significant shift in emotional processing towards positive words and reduced negative bias in assessing negative facial expressions. Effects on cognition were small and not statistically reliable and are not reported here. However, there was no evidence for any adverse effects. CONCLUSIONS: Consumption of a low dose of TRP-rich protein hydrolysate may have beneficial effects on emotional function that could promote feelings of wellbeing, possibly conferring resistance to deterioration in mood in healthy subjects or depressive episodes.

Proboscis lateralis.

INTRODUCTION: Proboscis lateralis is a rare congenitally acquired facial abnormality characterized by a soft-tissue tube- or trunk-like appendage projecting from the surface of the face, most frequently rooted in the medial canthal region. Proboscis lateralis is generally associated with a wide range of concomitant craniofacial anomalies, giving rise to multiple theories describing the embryological pathogenesis and various classification systems to account for the pathological associations. RESULTS/CONCLUSION: This paper provides a literature review of the rare manifestations of proboscis lateralis and represents a summary of current literature related to embryological pathogenesis, definitive diagnosis, and surgical management approaches.

Response of phosphomonoesterase activity in the lichen Cladonia portentosa to nitrogen and phosphorus enrichment in a field manipulation experiment.

*Effects of nitrogen (N) enrichment on the heathland lichen Cladonia portentosa were quantified to test the hypothesis that modified N : phosphorus (P) relationships observed in this species in N-polluted natural environments are a direct effect of increased N deposition, and to evaluate potential confounding effects of N form and P availability. *Cladonia portentosa was harvested from experimental plots in lichen-rich peatland vegetation (background total N deposition of 8 kg N ha(-1) yr(-1)) treated for 4 yr with additional wet N deposition at 0, 8, 24 and 56 kg N ha(-1) yr(-1) as either NH(4)(+) or NO(3)(-), and with or without P added at either 0.6 or 4 kg P ha(-1) yr(-1). *Nitrogen enrichment increased thallus N concentration, N : P mass ratio and phosphomonoesterase (PME) activity by factors of up to 1.3, 1.4 and 1.7, respectively, effects being independent of N form. Phosphomonoesterase activity was tightly related to thallus N : P ratio with additions of P at 4 kg ha(-1) yr(-1) depressing PME activity by a factor of 0.4. *Nitrogen enrichment induces P-limitation in C. portentosa with attendant changes in chemical and physiological characteristics that could be used as sensitive biomarkers with which to detect low levels of N pollution.

Effects of nitrogen enrichment on phosphatase activity and nitrogen : phosphorus relationships in Cladonia portentosa.

*Relationships between nitrogen deposition in the UK and phosphomonoesterase (PME) activity and nitrogen (N) and phosphorus (P) concentrations in Cladonia portentosa were quantified to understand factors limiting lichen growth and to further develop biomarkers for N pollution. *Lichen was collected from sites differing either in rates of wet N (NH(4)(+) + NO(3)(-)) deposition or in annual mean N concentration in rainfall based on both measured and modelled data sets. The PME activity, and total N and P concentrations were measured in specific horizontal strata in lichen mats and PME activity in the thallus was located using an enzyme-labelled fluorescent phosphatase substrate. *With an increase in modelled N deposition from 4.1 to 32.8 kg N ha(-1) yr(-1), PME activity, thallus N and N : P ratio increased by factors of 2.3, 1.4 and 1.8, respectively. Correlations with modelled data were generally stronger than with measured data and those with N deposition were stronger than those with N concentration in rainfall. The PME activity was located solely in the lichen fungus in outer regions of the thallus. *Nitrogen enrichment changes lichen N : P ratios from values typical of N limitation (for example, 10) to those indicative of P limitation (for example, 26) driving upregulation of PME activity.

Molecular and immunogenic features of myelin lipids: incitants or modulators of multiple sclerosis?

Myelin lipids have long been thought to play intriguing roles in the pathogenesis of multiple sclerosis (MS). This review summarizes current understanding of the molecular basis of MS with emphasis on the: (i.) physico-chemical properties, organization and accessibility of the lipids and their distribution within the myelin multilayer; (ii.) characterization of myelin lipid structures, and structure-function relationships relevant to MS mechanisms, and; (iii.) immunogenic and other features of lipids in MS including molecular mimicry, lipid enzyme genetic knockouts, glycolipid-reactive NKT cells, and monoclonal antibody-induced remyelination. New findings associate anti-lipid antibodies with pathophysiological biomarkers and suggest clinical utility. The structure of CD1d-lipid complexed with the lipophilic invariant T cell receptor (iTCR) may be crucial to understanding MS pathogenesis, and design of lipid antigen-specific therapeutics. Novel immuno-modulatory tools for treatment of autoimmune diseases including MS in which there is both constraint of inflammation and stimulation of remyelination are now emerging.

Maternal alcohol consumption increases sphingosine levels in the brains of progeny mice.

The effect of 'binge' alcohol upon sphingolipid metabolism in the fetal alcohol syndrome (FAS) was examined in pregnant mice (C57BL/6J) by administering a single dose of alcohol during the third trimester (gestational day 15-16). The control mice were administered a sucrose solution of equal caloric value. Brains from progeny at postnatal days 5, 15, 21 and 30 were dissected into three regions, and sphingolipid concentrations of the brain regions were determined including assay of monoglycosylceramide, ceramide, sphingosine and sphingomyelin. We found that a single dose of ethanol induces an elevation of sphingosine (2-3.5-fold) in the brain of progeny. The level of brain ceramide at a dose of 1.5 g/kg was significantly higher than control. Alcohol consumption during pregnancy induces neuronal loss in progeny brains. Our result suggests that the elevation of sphingosine in progeny brain induced by maternal alcohol consumption may be responsible for observed neuronal loss in FAS.

Safety and efficacy of an infectious bronchitis virus used for chicken embryo vaccination.

Commercial vaccines for in ovo vaccination have not yet been developed for infectious bronchitis virus (IBV), the major coronavirus in the poultry industry. Recombinant IBVs based on the Beaudette strain expressing the Beaudette spike protein (Beau-R) or that from the virulent M41 strain (BeauR-M41(S)) were assessed for their potential as prototype vaccines for application to 18-day-old embryos. Pathogenicity was assessed by observing the effect on hatchability, and/or the production of nasal discharge and/or the effects on ciliary activity in the trachea at various time points post hatch. In contrast to commercial IBV vaccines given in ovo, the Beau-R and BeauR-M41(S) strains did not reduce hatchability or cause nasal discharge, and caused minimal damage to the ciliated epithelium of the trachea. The presence of the spike protein from a virulent virus did not increase the pathogenicity of the virus according to the criteria used. Assessment of the BeauR-M41(S) strain for efficacy showed that it protected up to 90% of chicks against challenge with virulent IB virus (M41) in a dose dependent manner. Further egg passage of the BeauR-M41(S) strain (BeauR-M41(S) EP10) did not increase its pathogenicity though it did improve its efficacy, based on serology and protection against a virulent challenge. BeauR-M41(S) EP10 was more efficacious than BeauR-M41(S) protecting more birds against virulent challenge and providing a better serological antibody response. BeauR-M41(S) EP10 induced a serological response similar to that of a commercial vaccine given at day-old though the commercial vaccine provided slightly higher efficacy. These results are promising for the development of embryo safe efficacious IBV vaccines for in ovo application.

Relatively low levels of calpain expression in juvenile rat correlate with less neuronal apoptosis after spinal cord injury.

Approximately 5% of spinal cord injuries in the US occur in patients younger than 16 years. These young patients have an increased mortality within the 24 h after trauma but have a greater capacity for functional recovery than adults, suggesting age-related differences in injury tolerance. Unfortunately, the response of the developing cord to secondary injury has not been thoroughly investigated. Calpain, a Ca(2+)-dependent protease, has been implicated in the pathogenesis of spinal cord injury (SCI) in rats. Our current investigation revealed that following SCI, calpain upregulation was qualitatively less in the 21-day-old rats than in adult rats, as shown by immunofluorescent labeling. Decreased levels of TUNEL+ neurons were also noted in juvenile rat spinal cord, indicating that the developing cord may have an increased resistance to injury.

Inhibition of calpain-mediated apoptosis by E-64 d-reduced immediate early gene (IEG) expression and reactive astrogliosis in the lesion and penumbra following spinal cord injury in rats.

Upregulation of calpain, a Ca(2+)-activated cysteine protease, has been implicated in apoptosis and tissue degeneration in spinal cord injury (SCI) that over time spreads from the site of injury to the surrounding regions. We examined calpain content and activity, regulation of immediate early genes (IEGs) such as c-jun and c-fos, reactive astrogliosis as the expression of glial fibrillary acidic protein (GFAP), and apoptosis-related features such as caspase-3 mRNA expression and internucleosomal DNA fragmentation in 1-cm long spinal cord segments (S1, distant rostral; S2, adjacent rostral; S3, lesion or injury; S4, adjacent caudal; and S5, distant caudal) following SCI in rats. Calpain content and production of 150 kD calpain-cleaved alpha-fodrin fragment, expression of IEGs, reactive astrogliosis, and apoptotic features were highly increased in the lesion (S3), moderately in adjacent areas (S2 and S4), and slightly in distant areas (S1 and S5) in SCI rats when compared to sham animals. Administration of the calpain-specific inhibitor E-64-d (1 mg/kg) to SCI rats continuously for 24 h inhibited calpain activity and other factors contributing to apoptosis in the lesion and surrounding areas, indicating that calpain played a key role in the pathophysiology of SCI. The results obtained from this animal model of SCI suggest that calpain inhibitor can provide neuroprotection in patients with SCI.

Inhibition of K/HCO(3) cotransport in squid axons by quaternary ammonium ions.

Previous squid-axon studies identified a novel K/HCO3 cotransporter that is insensitive to disulfonic stilbene derivatives. This cotransporter presumably responds to intracellular alkali loads by moving K(+) and HCO(3)(-) out of the cell, tending to lower intracellular pH (pH(i)). With an inwardly directed K/HCO(3) gradient, the cotransporter mediates a net uptake of alkali (i.e., K(+) and HCO(3)(-) influx). Here we test the hypothesis that intracellular quaternary ammonium ions (QA(+)) inhibit the inwardly directed cotransporter by interacting at the intracellular K(+) site. We computed the equivalent HCO(3)(-) influx (J(HCO3)) mediated by the cotransporter from the rate of pH(i) increase, as measured with pH-sensitive microelectrodes. We dialyzed axons to pH(i) 8.0, using a dialysis fluid (DF) free of K(+), Na(+) and Cl(-). Our standard artificial seawater (ASW) also lacked Na(+), K(+) and Cl(-). After halting dialysis, we introduced an ASW containing 437 mm K(+) and 0.5% CO(2)/12 mm HCO(3)(-), which (i) caused membrane potential to become transiently very positive, and (ii) caused a rapid pHi decrease, due to CO(2) influx, followed by a slower plateau-phase pH(i) increase, due to inward cotransport of K(+) and HCO(3)(-). With no QA(+) in the DF, J(HCO3) was approximately 58 pmole cm(-2) sec(-1). With 400 mm tetraethylammonium (TEA(+)) in the DF, J(HCO3) was virtually zero. The apparent K(i) for intracellular TEA(+) was approximately 78 mm, more than two orders of magnitude greater than that obtained by others for inhibition of K(+) channels. Introducing 100 mm inhibitor into the DF reduced J(HCO3) to approximately 20 pmole cm(-2) sec(-1) for tetramethylammonium (TMA(+)), approximately 24 for TEA(+), approximately 10 for tetrapropylammonium (TPA(+)), and virtually zero for tetrabutylammonium (TBA(+)). The apparent K(i) value for TBA(+) is approximately 0.86 mm. The most potent inhibitor was phenyl-propyltetraethylammonium (PPTEA(+)), with an apparent K(i) of approximately 91 microm. Thus, trans-side quaternary ammonium ions inhibit K/HCO(3) influx in the potency sequence PPTEA(+) > TBA(+) > TPA(+) > TEA(+) congruent with TMA(+). The identification of inhibitors of the K/HCO(3) cotransporter, for which no inhibitors previously existed, will facilitate the study of this transporter.

Cell death in spinal cord injury (SCI) requires de novo protein synthesis. Calpain inhibitor E-64-d provides neuroprotection in SCI lesion and penumbra.

Degradation of cytoskeletal proteins by calpain, a Ca(2+)-dependent cysteine protease, may promote neuronal apoptosis in the lesion and surrounding areas following spinal cord injury (SCI). Clinically relevant moderate (40 g-cm force) SCI in rats was induced at T12 by a standardized weight-drop method. Internucleosomal DNA fragmentation or apoptosis in the lesion was inhibited by 24-h treatment of SCI rats with cycloheximide (1 mg/kg), indicating a requirement for de novo protein synthesis in this process. To prove an involvement of calpain activity in mediation of apoptosis in SCI, we treated SCI rats with a cell-permeable calpain inhibitor E-64-d (1 mg/kg). Following 24-h treatment, a 5-cm-long spinal cord section centered at the lesion was collected, and divided equally into five segments (1 cm each) to determine calpain activity, as shown by degradation of the 68-kD neurofilament protein (NFP), and apoptosis as indicated by internucleosomal DNA fragmentation. Neurodegeneration propagated from the site of injury to neighboring rostral and caudal regions. Both calpain activity and apoptosis were readily detectable in the lesion, and moderately so in neighboring areas of untreated SCI rats, whereas these were almost undetectable in E-64-d-treated SCI rats, and absent in sham animals. Results indicate that apoptosis in the SCI lesion and penumbra is prominently associated with calpain activity and is inhibited by the calpain inhibitor E-64-d providing neuroprotective benefit.

Identification and expression analysis of spastin gene mutations in hereditary spastic paraplegia.

Pure hereditary spastic paraplegia (SPG) type 4 is the most common form of autosomal dominant hereditary SPG, a neurodegenerative disease characterized primarily by hyperreflexia and progressive spasticity of the lower limbs. It is caused by mutations in the gene encoding spastin, a member of the AAA family of ATPases. We have screened the spastin gene for mutations in 15 families consistent with linkage to the spastin gene locus, SPG4, and have identified 11 mutations, 10 of which are novel. Five of the mutations identified are in noninvariant splice-junction sequences. Reverse transcription-PCR analysis of mRNA from patients shows that each of these five mutations results in aberrant splicing. One mutation was found to be "leaky," or partially penetrant; that is, the mutant allele produced both mutant (skipped exon) and wild-type (full-length) transcripts. This phenomenon was reproduced in in vitro splicing experiments, with a minigene splicing-vector construct only in the context of the endogenous splice junctions flanking the splice junctions of the skipped exon. In the absence of endogenous splice junctions, only mutant transcript was detected. The existence of at least one leaky mutation suggests that relatively small differences in the level of wild-type spastin expression can have significant functional consequences. This may account, at least in part, for the wide ranges in age at onset, symptom severity, and rate of symptom progression that have been reported to occur both among and within families with SPG linked to SPG4. In addition, these results suggest caution in the interpretation of data solely obtained with minigene constructs to study the effects of sequence variation on splicing. The lack of full genomic sequence context in these constructs can mask important functional consequences of the mutation.

Chromatographic resolution and quantitative assay of CNS tissue sphingoids and sphingolipids.

Quantitative separation of ceramide, sphingoids (dihydrosphingosine, sphingosine, psychosine), and glycosphingolipids as individual fractions was achieved with silicic acid, Dowex column chromatography, and specific solvent mixtures that have not been previously described. Purified ceramide, resolved as a single band, was assayed by thin-layer chromatography (TLC) followed by gas chromatography (GC) and high performance liquid chromatography (HPLC). Sphingoids, purified by Dowex, were assayed by GC and HPLC without mild alkaline hydrolysis, which reduces the yield by interfering with the free amino group of psychosine and dihydrosphingosine. Several less polar (than cerebroside) alkali-/acid-labile glycosphingolipids that elute with galactosylceramide were also identified. Neutral and acidic glycosphingolipids, quantitatively recovered and purified to homogeneity, were resolved by TLC. We used these techniques to determine sphingolipids and sphingoids of vertebrate central nervous system (CNS) tissue, using as little as 30-50 mg (wet weight) of tissue. In addition, phosphatidylcholine and sphingomyelin, relevant to ceramide metabolism, were quantitatively recovered in pure form and resolved by TLC. This method, used to study CNS sphingolipid content, may well be applicable to determine the sphingolipid composition of other tissues and cell culture, but further experiments are necessary to ascertain this.