Molecular autopsy for fetal structural anomaly: diagnostic and clinical utility of multidisciplinary team approach.

OBJECTIVES: In the West Midlands Regional Genetic Service, cases of perinatal death with a possible genetic diagnosis are evaluated by the Perinatal Pathology Genetic Multidisciplinary Team (MDT). The MDT assessed autopsy findings and considered genomic assessments. The objective of this retrospective service evaluation was to determine the clinical utility of the MDT. This is the first evaluation since the introduction of whole genome and whole exome sequencing in routine clinical care. METHOD: The outcomes for all the perinatal MDT cases from January 2021 to December 2021 were examined. All cases received a full or partial post-mortem examination (PM) and a chromosomal microarray. Demographics, phenotype, MDT recommendations, genetic testing, diagnoses, outcomes, impact of PM and impact of genetic testing were collected from patient case notes. RESULTS: One hundred and twenty-three cases were discussed at the MDT meeting in 2021. Genetic evaluation was recommended in 84 cases and accepted in 64 cases. A range of genetic tests were requested according to indication and availability. Thirty diagnoses were identified in 29 cases from 26 unrelated families. The diagnostic yield was 24% (29/123) of all cases or 45% (29/64) of the cases with a suspected genetic diagnosis who underwent genetic testing. PM examination added clinically actionable phenotype data in 79% of cases. A genetic diagnosis enabled accurate counselling of recurrence risk and provision of appropriate follow-up, including prenatal testing and preimplantation diagnosis for patients with inherited conditions. CONCLUSIONS: Genomic testing was a clinically useful addition to (but not a substitute for) PM examination in perinatal cases associated with structural anomalies. The MDT model helped assess cases and plan appropriate follow-up. Expedited whole genome sequencing or panel-agnostic analysis were most appropriate for heterogeneous presentations. This broad approach can also expand prenatal phenotypes and detect novel disease genes and should be a priority for future research. This article is protected by copyright. All rights reserved.

Laparoscopic repair of postoperative perineal hernia using a two-mesh technique.

AIM: Perineal herniation following abdomino-perineal excision of the rectum (APER) can be debilitating. Repair options include a transabdominal (laparoscopic or open), perineal or a combined approach, but there is no consensus on the optimal technique. We describe a novel laparoscopic two-mesh technique and short- to medium-term outcomes. METHOD: Six patients underwent this operation between 2008 and 2014. Patients were positioned in a modified Lloyd-Davies position, allowing perineal access, and steep Trendelenburg to aid displacement of small bowel from the pelvis. A polypropylene mesh was shaped, placed over the hernial defect, tacked postero-laterally and sutured antero-laterally to reconstitute the pelvic diaphragm. A second larger mesh (composite) was placed over the first supporting mesh and secured with tacks and sutures, overlapping the hernial defect, preventing small bowel contact with the mesh. RESULTS: The median time from the index operation to presentation of the hernia was 5 months. One patient with dense small bowel adhesions from the primary repair had a combined laparoscopic and perineal approach. The median operating time was 141 min and median length of stay was 3 days. There were no intra-operative complications and no recurrences over a follow-up of 1-76 months. CONCLUSION: We describe a novel laparoscopic technique for perineal hernia repair following APER with a low recurrence rate in the intermediate term.

Rapid prenatal diagnosis of common trisomies: discordant results between QF-PCR analysis and karyotype analysis on long-term culture for a case of trisomy 18 detected in CVS.

OBJECTIVES: QF-PCR analysis can be used as a rapid test to diagnose primary trisomy in prenatal samples. Mosaicism in CVS detected by QF-PCR has previously been reported; however, no case has so far been reported in which the QF-PCR result was completely discrepant to that of the karyotype analysis from a long-term culture. METHODS: A CVS, referred because of a high serum screening risk of 1:10 for Down Syndrome and 1:110 for Edwards Syndrome, was tested by QF-PCR analysis and chromosome analysis of cultured cells. Subsequent analyses were carried out on a follow-up amniotic fluid sample and foetal tissue samples. RESULTS: Conflicting results were obtained between QF-PCR analysis on two independent fronds from the chorionic villi and chromosome analysis on cultured CVS. Cytogenetic and molecular analysis on a subsequent amniotic fluid sample indicated trisomy 18 with no evidence of mosaicism. Analysis of follow-up tissue confirmed trisomy in a foetal skin sample and mosaicism for trisomy 18 in four placental sites tested. CONCLUSION: We report here an apparently normal CVS QF-PCR result that was completely discrepant with the trisomy 18 positive karyotype result on long-term culture. This has important implications regarding our current testing protocol.

Microsatellite marker development and analysis in the eastern oyster (Crassostrea virginica): confirmation of null alleles and non-Mendelian segregation ratios.

Eighteen microsatellite markers were developed for the Crassostrea virginica nuclear genome, including di-, tri-, and tetranucleotide microsatellite repeat regions that included perfect, imperfect, and compound repeat sequences. A reference panel with DNA from the parents and four progeny of 10 full-sib families was used for a preliminary confirmation of polymorphism at these loci and indications of null alleles. Null alleles were discovered at three loci; in two instances, primer redesign enabled their amplification. Two to five representative alleles from each locus were sequenced to ensure that the targeted loci were amplifying. The sequence analysis revealed not only variation in the number of simple sequence repeat units, but also polymorphisms in the microsatellite flanking regions. A total of 3626 bp of combined microsatellite flanking region from the 18 loci was examined, revealing indels as well as nucleotide site substitutions. Overall, 16 indels and 146 substitutions were found with an average of 4.5% polymorphism across all loci. Eight markers were tested on the parents and 39-61 progeny from each of four families for examination of allelic inheritance patterns and genotypic ratios. Twenty-six tests of segregation ratios revealed eight significant departures from expected Mendelian ratios, three of which remained significant after correction for multiple tests. Deviations were observed in both the directions of heterozygote excess and deficiency.

Exoskeleton for forearm pronation and supination rehabilitation.

Loss of function after SCI, ABI or stroke has a marked affect on ones quality of life. Return of function has been a long-standing goal of physical and occupational therapy. Repeated motor practice has been identified as crucial for motor recovery. The development of a robotic device for neuromotor rehabilitation and upper extremity neuromuscular system recovery is described. The actuator mechanism allows free motion when possible, and provides programmable therapeutic levels of resistance. The sensor system allows characterization of the applied forces, and accurate measurement of the range of motion of the joint. The control system provides real time feedback of actuator commands based on sensor data, calibration routines, and operational modes.

Spatial partitioning of host habitat by chewing lice of the genera Geomydoecus and Thomomydoecus (Phthiraptera: Trichodectidae).

Chewing lice, Geomydoecus and Thomomydoecus, coexist on pocket gophers, Thomomys spp. We investigated the spatial distribution of the 2 genera on their hosts and explored possible mechanisms of resource partitioning by chewing lice. Chewing lice appear to partition available host resources spatially, with Geomydoecus occurring primarily on the lateral and dorsal regions of the host, and Thomomydoecus occurring primarily on the lateral and ventral regions. Although spatial partitioning of the host habitat is evident, it does not appear to be explained by hair diameter. Spatial partitioning of the host's body could be the result of some other factor, possibly temperature or humidity gradients of the host's body.

2-Nitrobenzaldehyde: a convenient UV-A and UV-B chemical actinometer for drug photostability testing.

We report the development of a new 'photochemical titration' actinometric method for measurement of UV-B (290-320 nm) and UV-A (320-400 nm) light dose during drug photostability testing. It is based upon photolysis of aqueous 2-nitrobenzaldehyde solution, a well-characterized reaction that has been previously demonstrated to be useful as an accurate and reliable actinometric method. Our new method can be performed by a chemical technician using only common reagents. It has been developed for use with xenon arc lamp illumination chambers which are commonly employed for drug photostability testing, but can be readily modified for use with the other illumination sources.

Photochemical formation of singlet molecular oxygen ((1)O2) in illuminated 6-methylcoumarin solutions.

Use of the fragrance 6-methylcoumarin (6-MC) in cosmetic products has declined significantly due to numerous reports of photoallergic contact dermatitis associated with its use. We have determined that 6-MC undergoes direct photolysis with an estimated half-life of 83 minutes when illuminated with mid-latitude U.S., noon-centered, equinox sunlight and a quantum yield for photolysis at 313 nm of phi = 3 x 10(-3). The work presented here also provides evidence that singlet molecular oxygen ((1)O2) is formed in illuminated solutions containing 6-MC. An estimated value of phi = 0.01 is reported for the (1)O2 quantum yield at 313 nm. Formation of (1)O2 is significant because it is known to react with a variety of biomolecules and it is possible that (1)O2 formation is at least partially responsible for reports of 6-MC photoallergenicity and phototoxicity.

Sex and meiosis in autotetraploid Pacific oyster, Crassostrea gigas (Thunberg).

Sex and meiosis were studied in induced autotetraploids of the Pacific oyster (Crassostrea gigas Thunberg) and were compared with sex and meiosis in autotriploids and normal diploids. Tetraploid oysters reached sexual maturity at 1 year of age in an approximately 1:1 sex ration. In contrast with the abnormally high frequency of hermaphrodites among triploids, tetraploids had about the same level of hermaphrodites as normal diploids. Fecundity of tetraploids was comparable to that of normal diploids, differing from the greatly reduced fecundity of triploids. Homologous chromosomes synapsed predominantly as trivalents in eggs from triploids and as quadrivalents in eggs from tetraploids. After fertilization, eggs from tetraploids and triploids went through two meiotic divisions, as normal eggs did. The average gamete chromosome number was 10.0 for diploids and 19.9 for tetraploids. The distribution of gamete chromosome numbers from triploids suggested that the extra chromosome in the trivalent segregated randomly during anaphase I. In tetraploids, however, the two extra chromosomes in the quadrivalents did not segregate independently and, instead, they preferentially cosegregated to opposite poles producing balanced gametes. These results suggest that mechanisms may exist to weight, balance, and equally distribute quadrivalents, possibly through mitotic force and tension. Errors in chromosome balancing in normal meiosis may result in nondisjunction, which is the primary cause of human aneuploidy.

Chromosome Segregation in Fertilized Eggs From Triploid Pacific Oysters, Crassostrea gigas (Thunberg), Following Inhibition of Polar Body 1.

Chromosome segregation in fertilized eggs from triploid Pacific oysters, following inhibition of the first polar body (PB1), was studied with acetic orcein staining techniques. To block the release of PB1, fertilized eggs were treated with 0.5 mg/l of cytochalasin B (CB). Four types of segregation were observed, namely, "tripolar segregation" (54.5%), "united bipolar segregation" (12%), "separated bipolar segregation" (2.5%), and "incomplete united bipolar segregation" (4%). The remaining 23% could not be classified because of chromosome disorganization, but appeared to be variants of the above. It seemed clear that the predominant pattern that gave rise to tetraploids was united bipolar segregation, although certain separated bipolar segregations might also lead to the formation of tetraploids. The sequential events of meioses observed in CB-treated eggs are described. The asynchrony of meiotic events and possible mechanisms for the various types of chromosome segregation are discussed.

Complete interference and nonrandom distribution of meiotic crossover in a mollusc, Mulinia lateralis (Say).

In gene mapping, the genetic distance between two genes is measured by the frequency of meiotic crossovers occurring between them. It is generally assumed that there is more than one crossover per chromosome, and the distribution of crossovers along chromosomes is random and follows a Poisson distribution (no interference), or that interference is inversely correlated with distance. Although those assumptions may be correct for some organisms, we report here a novel exception in the marine mollusc Mulinia lateralis Say. Using segregation analysis of gynogenetic diploids, we found surprisingly high gene-centromere recombinant frequencies for most of the 13 allozyme loci studied. For at least six loci, there was always one and only one crossover occurring between the gene and its centromere, suggesting complete interference where the occurrence of one crossover completely suppressed the occurrence of another. The complete interference was confirmed by the cytogenetic observation that there was only one chiasma for all bivalents. Further, sites of the single crossover seem not to be randomly distributed along chromosomes, but preferentially located in a recombination hot-region proximal to the centromere. The restricted distribution of a single crossover per chromosome provides one explanation for the unique phenomenon of heterozygote deficiency in M. lateralis and other molluscs.

Photochemical formation of singlet molecular oxygen in illuminated aqueous solutions of several commercially available sunscreen active ingredients.

Evidence is presented for the photochemical formation of singlet molecular oxygen (1O2) in air-saturated aqueous solutions of several sunscreen active ingredients using sunlight-range illumination. This is of significance because (1) 1O2 is known to be cytotoxic, and (2) there have been several reports of toxic effects associated with the use of some sunscreens; most notably, with p-aminobenzoic acid (PABA). Illuminated aqueous solutions of PABA, 2-ethylhexyl p-(dimethylamino)benzate (ODPABA), 2-hydroxy-4-methoxybenzophenone (BZ3), 2,2'-dihydroxy-4-methoxybenzophenone (BZ8), 2-ethylhexyl 2-cyano-3,3-diphenylacrylate (OCR), 2-ethylhexyl p-methoxycinnamate (OMC), and 2-ethylhexyl salicylate (OCS) were evaluated individually for 1O2 formation. Furfuryl alcohol (FFA), a well-known chemical trap for 1O2, was added to each of the aqueous sunscreen solutions. The FFA was consumed when solutions of PABA, ODPABA, OMC, and OCR were illuminated, but no loss of FFA other than by direct photolysis occurred in solutions of BZ3, BZ8, or OCS. There was also no significant loss of FFA in any of these solutions kept in the dark. Further evidence for the formation of 1O2 in illuminated aqueous sunscreen solutions is provided by the results of experiments in which individual solutions containing sunscreen active ingredients and FFA that were diluted with D2O exhibited an increased rate of FFA consumption while the addition of azide ion (N3-) reduced the rate of FFA consumption. Continuous sunlight-range illumination of aqueous PABA solutions produced significantly higher steady-state concentrations of 1O2 than in solutions containing any of the other sunscreen active ingredients evaluated. The substituted benzophenone compounds (BZ3 and BZ8) and the salicylate-based compound (OCS) not only appear to produce no 1O2, but they also appear to produce no other reactive oxidant species that are capable of consuming FFA. This indicates that BZ3, BZ8, and OCS may be peferable, from the standpoint of toxic oxidant formation, for use as sunscreen active ingredients when compared to the other compounds evaluated in this study.

Production of transgenic dwarf surfclams, Mulinia lateralis, with pantropic retroviral vectors.

A pantropic pseudotyped retroviral vector containing the envelope protein of vesicular stomatitis virus was used as a gene transfer vector in the dwarf surfclam, Mulinia lateralis. These pantropic retroviral vectors have an extremely broad host cell range and can infect many nonmammalian species. Newly fertilized dwarf surfclam eggs were electroporated at 700 V in the presence of 1 x 10(4) colony-forming units of pantropic pseudotyped retroviral particles. Infection was well tolerated and did not affect the survival rate of the embryos. Gametes collected from P1 presumptive transgenic animals were analyzed for the presence of provirus by PCR, and in different experiments 13-33% of the gamete pools were positive for the transgene. Dot blot hybridization of DNA samples from the F1 offspring of two different crosses between infected P1 and wild-type individuals revealed that 28% and 31% of F1 offspring were transgenic, respectively. Southern blot analysis of DNA isolated from PCR-positive F1 animals confirmed integration of a single copy of the provirus into the host genome. Thus, the germ lines of these two P1 transgenic animals were mosaic for the transgene. Expression of beta-galactosidase encoded by the provirus was detected in transgenic but not control surfclam embryos. Pantropic pseudotyped retroviral vectors provide a useful method for the stable introduction of foreign genetic information into surfclams and may facilitate the introduction of desirable genetic traits into commercially important shellfish and crustaceans.

Rapid reaction of singlet molecular oxygen (1O2) with p-aminobenzoic acid (PABA) in aqueous solution.

The reaction of singlet molecular oxygen (1O2) with p-aminobenzoic acid (PABA) is quite rapid. The rate constant for reaction of 1O2 with PABA was measured in buffered aqueous solution by the method of competition kinetics and found to be 8.9 x 10(8) M-1 sec-1. The results of a Stern-Volmer analysis indicate that additional loss of 1O2 does not occur through physical quenching by PABA. Although illuminated PABA solutions are known to form 1O2, it appears that PABA is also a very efficient sink for 1O2.

Sex determination and polyploid gigantism in the dwarf surfclam (Mulinia lateralis Say).

Mulinia lateralis, the dwarf surfclam, is a suitable model for bivalve genetics because it is hardy and has a short generation time. In this study, gynogenetic and triploid M. lateralis were successfully induced. For gynogenesis, eggs were fertilized with sperm irradiated with ultraviolet light and subsequently treated with cytochalasin B to block the release of the second polar body (PB2). Triploidy was induced by blocking PB2 in normally fertilized eggs. The survival of gynogenetic diploids was very low, only 0.7% to 8 days post-fertilization (PF), compared with 15.2% in the triploid groups and 27.5% in the normal diploid control. Larvae in all groups metamorphosed at 8-10 days PF, and there was no significant post-larval mortality. At sexual maturation (2-3 months PF), all gynogenetic diploids were female, and there was no significant difference (P > 0.05) in sex ratio between diploids and triploids. These results suggested that the dwarf surfclam may have an XX-female, XY-male sex determination with Y-domination. Compared with diploids, triploids had a relative fecundity of 59% for females and 80% for males. Eggs produced by triploid females were 53% larger (P < 0.001) in volume than those from diploid females. In both length and weight measurements at three months PF, the gynogenetic diploids were not significantly (P > 0.33) different from normal diploid females, suggesting that inbreeding depression was minimal in meiosis II gynogens. Triploid clams were significantly larger (P < 0.001) than normal diploids. We hypothesize that the increased body-size in triploids was caused by a polyploid gigantism due to the increased cell volume and a lack of cell-number compensation.

Reproductive Potential and Genetics of Triploid Pacific Oysters, Crassostrea gigas (Thunberg).

The reproductive potential and genetics of triploidy were studied in the Pacific oyster. DNA content in sperm from triploids showed a single peak at 1.5c as determined by flow cytometry. In eggs from triploids, trivalents were the dominant form of synapsed chromosomes, although the degree of synapsis varied considerably within and among females. Some eggs went through complete synapsis and formed 10 trivalents, chromosomes; most had a mixture of 11-13 trivalents, bivalents, and univalents. Factorial matings were produced from diploid (D) and triploid (T) parent oysters, creating four crosses: DD, DT, TD, and TT (female first). Gametes from triploids were fully capable of fertilization. After fertilization, eggs from triploids went through two meioses and released two polar bodies as diploid eggs did. Karyological analyses showed that average ploidy of the resultant embryos was 2.0 n for DD, 2.46 n for DT, 2.52 n for TD, and 2.88 n for TT. Survival of fertilized eggs to metamorphosis and settlement was about 21% for DD, but considerably lower on other crosses: 0.0007% for DT, 0.0463% for TD, and 0.0085% for TT. Nine months after matings, all survivors from DT crosses were diploid. Survivors from TD crosses consisted of 33% diploids, 57% triploids, and 10% tetraploids. Survivors from the TT crosses consisted of 90% triploids, 4% diploids, and 6% mosaics. We hypothesize that differences in ploidy composition between DT and TD embryos and survivors were caused by pro-egg segregations that favor the retention, rather than loss, of extra chromosomes in the egg. The reproductive potential of triploids and evolutionary implications are discussed.

Selection and implementation of an automated care planning system for a health care institution.

This article describes the process of selecting and implementing an automated care planning system in a 634-bed hospital setting. Obstacles to system selection and personnel training are discussed. The advantages and disadvantages of automated systems as they impact nursing staff are delineated. Recommendations for nursing managers interested in selecting and using automated care planning systems are outlined.