Chromosome-level genome sequence of the Genetically Improved Farmed Tilapia (GIFT, Oreochromis niloticus) highlights regions of introgression with O. mossambicus.

BACKGROUND: The Nile tilapia (Oreochromis niloticus) is the third most important freshwater fish for aquaculture. Its success is directly linked to continuous breeding efforts focusing on production traits such as growth rate and weight. Among those elite strains, the Genetically Improved Farmed Tilapia (GIFT) programme initiated by WorldFish is now distributed worldwide. To accelerate the development of the GIFT strain through genomic selection, a high-quality reference genome is necessary. RESULTS: Using a combination of short (10X Genomics) and long read (PacBio HiFi, PacBio CLR) sequencing and a genetic map for the GIFT strain, we generated a chromosome level genome assembly for the GIFT. Using genomes of two closely related species (O. mossambicus, O. aureus), we characterised the extent of introgression between these species and O. niloticus that has occurred during the breeding process. Over 11 Mb of O. mossambicus genomic material could be identified within the GIFT genome, including genes associated with immunity but also with traits of interest such as growth rate. CONCLUSION: Because of the breeding history of elite strains, current reference genomes might not be the most suitable to support further studies into the GIFT strain. We generated a chromosome level assembly of the GIFT strain, characterising its mixed origins, and the potential contributions of introgressed regions to selected traits.

Norwich COVID-19 testing initiative pilot: evaluating the feasibility of asymptomatic testing on a university campus.

BACKGROUND: There is a high prevalence of COVID-19 in university-age students, who are returning to campuses. There is little evidence regarding the feasibility of universal, asymptomatic testing to help control outbreaks in this population. This study aimed to pilot mass COVID-19 testing on a university research park, to assess the feasibility and acceptability of scaling up testing to all staff and students. METHODS: This was a cross-sectional feasibility study on a university research park in the East of England. All staff and students (5625) were eligible to participate. All participants were offered four PCR swabs, which they self-administered over two weeks. Outcome measures included uptake, drop-out rate, positivity rates, participant acceptability measures, laboratory processing measures, data collection and management measures. RESULTS: 798 (76%) of 1053 who registered provided at least one swab; 687 (86%) provided all four; 792 (99%) of 798 who submitted at least one swab had all negative results and 6 participants had one inconclusive result. There were no positive results. 458 (57%) of 798 participants responded to a post-testing survey, demonstrating a mean acceptability score of 4.51/5, with five being the most positive. CONCLUSIONS: Repeated self-testing for COVID-19 using PCR is feasible and acceptable to a university population.

"Support for the supporters": a qualitative study of the use of WhatsApp by and for mentor mothers with HIV in the UK.

Third-sector organizations, an important support for people living with HIV, increasingly use digital technology to improve service efficiency and reach. However, there is limited empirical evidence on this use by women living with HIV. The 4M Network (4MNet) is a peer-run UK-wide network of trained Mentor Mothers (MMs) living with HIV; it uses the WhatsApp platform as its primary digital communication tool. We report on a qualitative study about 4MNet MMs' experiences of using WhatsApp, to inform the design of future digital support services. Seven telephone interviews were conducted with five MMs and two project management team (PMT) members in February 2019. Interviews were analyzed using Interpretive Phenomenological Analysis (IPA). WhatsApp was found to have several key features that provided both positive and negative use considerations. WhatsApp eased communication among MMs and supported participation in group activities despite differing schedules and geographic locations. Challenges encountered with WhatsApp included: financial restrictions to data storage and continual access; self-confidence using technology; and security and privacy concerns. Peer-led digital communication is found to be acceptable and effective for women living with HIV. Understanding barriers and valued features of existing digital platforms increasingly used among potentially marginalized groups is vital for informing inclusive innovation.

Numerical modelling and CFD simulation of a polymer electrolyte membrane (PEM) fuel cell flow channel using an open pore cellular foam material.

Fuel cell performances vary with different structural configurations and materials. However, the two main areas that determine this performance metric are the membrane electrode assembly (MEA) and the bipolar plates. The MEA provides the platform for the electrochemical reaction to occur and the bipolar plate serves as a medium between the reactants (hydrogen and air) and the catalyst layer. The bipolar plate is the first point of contact for the reactants inside the fuel cell, so a badly designed item with a high pressure drop will have a negative impact on fuel cell performance. Numerical modelling and simulation tools like ANSYS have a huge impact on engineering industry as they help designs to be validated and analysed before any physical construction. This investigation considers five suitable flow plate designs for PEM fuel cell, each completely different from the readily available, traditional serpentine designs on the market. The work explored the possibility of replacing these flow channels with an aluminium cellular foam with different inlet and outlet orientations. The designs were further optimised and modelled in ANSYS. The results obtained were compared with other designs in the literature. Compared to the serpentine flow design, the open pore cellular foam material showed a very small pressure drop in the range of 30-40 Pa. This indicates a possibility of replacing the traditional flow plate designs with the proposed ones.

Cholinesterases in development and disease.

Cholinesterases (ChEs) including acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) are abundant in the nervous system and other tissues. Here we describe two different aspects of ChEs and the cholinergic system. The first aspect concerns the role of cholinergic transmission in central pattern generation in the neonatal rat spinal cord and the second one describes the involvement of ChEs in the pathologies of dystrophin-deficient mutant (mdx) mice, the animal model of Duchenne muscular dystrophy. Thus, this study is divided into two distinct parts. In the first part we show that AChE is abundant in ventral horn neurons, central canal-adjacent and partition neurons in all the observed segments (L2, L5, S1, and S2). AChE was also found in the intermediolateral and sacral parasympathetic nuclei of L2 and S1, respectively. Blocking the AChE by edrophonium produced non-stationary bursting in spinal cord preparations of developing rats. Cross-wavelet/coherence analyses of the data revealed epochs of locomotor-like activity (left-right and flexor-extensor alternation) followed by other rhythmic or non-rhythmic bursting patterns. Addition of exogenous ACh stabilized the rhythm and increased the incidence of locomotor-like pattern in the preparations. Thus, the cholinergic system in the spinal cord is capable of producing and modulating functional rhythmic bursts. Moreover, bath-applied edrophonium and exogenous ACh were found as potent means of modulation of the locomotor rhythm produced by stimulation of sacrocaudal afferents (SCAs). We show that a subclass of sacral neurons with contralateral funicular projections to the thoracolumbar cord is associated with the cholinergic system. This group of neurons may play a major role in the observed enhancement of the SCA-induced motor rhythm. In the second part we show that adult mdx-muscles are malformed with distorted neuromuscular junctions (nmjs) and impaired regulation of acetylcholine receptors. Examination of circulating ChE levels revealed that in mdx-sera, while AChE activity was elevated, BuChE activity was markedly lower than in wild-type (wt) sera. Thus, BuChE to AChE ratio in mouse sera decreased from 6:1 in wt control to 3:1 in mdx. Because serum ChE levels may be modulated by gonadal steroids, it is possible that lack of dystrophin in mdx-mice may affect this regulation. Further studies are in progress to determine the potential endocrine regulation of ChEs in circulation and at the nmjs of mdx- and wt-mice. These studies will help clarify whether the hormonal regulation is impaired in the mdx mutant, and whether changes in circulating ChE reflect or influence the functional deficits observed in excitable tissues of diseased states.

From presentation to publication: the natural history of orthopaedic abstracts in the United Kingdom.

BACKGROUND: Up to 63% of the chapters in major orthopaedic textbooks use the results from abstracts that have been presented at international orthopaedic meetings. METHODS: Orthopaedic abstracts were reviewed that were presented at the 1997 and 1998 meetings of the British Orthopaedic Association and other specialist orthopaedic meetings. The number of abstracts that had gone on to a full text publication was assessed and changes in study design or outcome were determined. RESULTS: Of the 415 abstracts 137 (33.0%) went on to full text publication. Abstracts presented at the British Orthopaedic Association were significantly more likely to go on to full text publication than abstracts from the other meetings studied. The mean time to publication was 15.6 months. Sample sizes in unpublished studies were smaller (mean 129.8 subjects compared with a mean of 191.4 subjects for published studies). Of full text papers, 19.0% differed regarding study design from the abstract presented at the initial meeting and 10.9% had published different results. Randomised controlled trials had the highest rate of later full text publication (53.6%) followed by observational studies (32.8%), basic science studies (31.4%), and case reports (6.7%). CONCLUSIONS: In comparison with a study from North America, similar numbers of abstracts presented at meetings finally became published as full text articles, the abstracts had fewer authors, more often included randomised controlled trials and follow up data, and had fewer changes to the results. It is questionable whether the inclusion of such results from abstracts presented at international meetings by major orthopaedic textbooks should be undertaken before full text publication.

A study of the gonadotropin releasing hormone neuronal network in the median eminence of the rhesus monkey ( Macaca mulatta) using a post-embedding immunolabelling procedure.

The purpose of this study was to describe the ultrastructural features of gonadotropin releasing hormone (GnRH) axonal processes in the median eminence of the monkey, using a post-embedding immunogold labelling procedure. Evidence was also sought to evaluate the view that release of this peptide may be governed by direct inputs to GnRH axons in the median eminence. Plastic embedding was used to preserve ultrastructure, and a polyclonal rabbit anti-GnRH was used as primary antibody. Immunogold labelling with 15-nm particles was almost exclusively found overlying dense core vesicles (dcvs) and preabsorption of the primary antibody with synthetic GnRH eliminated this labelling. Morphometric analysis was performed on tissue from two monkeys. Four types of profiles containing GnRH immunoactive dcvs were observed. Type I profiles were morphologically unremarkable with a cross sectional area of approximately 0.6 microm2 and probably represent intervaricose axon segments. Type II profiles, which were nominally larger than Type I structures, were characterized by a high density of round microvesicles, which were frequently concentrated along the neuronal membrane to form 'synaptoid' contacts with adjacent glia. Two additional and large GnRH profiles (>5 microm2) were observed. One (Type III) contained a high density of dcvs and mitochondria, and was considered analogous to an axonal swelling or Herring body in the magnocellular hypothalamo-neurohypophysial system. The Type IV structure, which was considered not to be a Herring body because of the relative low density of mitochondria was innervated by a classical symmetrical synapse. The functional significance of these observations is discussed.

Development and organization of the hypophysiotropic hypothalamus driving the pituitary-gonadal axis in the rhesus monkey.

The purpose of the present review is to describe, with particular emphasis on the rhesus monkey, the ontogeny and functional organisation of the hypothalamic GnRH pulse generator. Control of pituitary-gonadal axis in higher primates is provided by a group of some 1,000 GnRH neurons that are diffusely distributed throughout the hypothalamus. After synthesis of a prehormone and formation of the mature decapeptide, GnRH is released in the hypophysial portal circulation and stimulates FSH and LH production.

Circulating leptin does not appear to provide a signal for triggering the initiation of puberty in the male rhesus monkey (Macaca mulatta).

The notion that circulating leptin may provide a somatic signal for timing the onset of puberty was examined in the male rhesus monkey. Circulating leptin levels were determined at weekly or biweekly intervals by RIA in intact (N=6) and prepubertally castrated monkeys (N=5) from approximately 18 to 30 months of age. Circulating testosterone (T) and gonadotropin levels were used as indices to identify the onset of the pubertal reaugmentation of pulsatile GnRH release (week 0) in intact and castrated animals, respectively. Time courses of the peripubertal changes in leptin concentrations in individual monkeys were normalized to week 0. In the intact group, mean leptin concentrations at this critical stage of development (week -26 to week +9) were unremarkable, ranging from 1.6+/-0.3 (SEM) to 2.4+/-0.6 ng/ml (P>0.05). In agonadal males, the pubertal onset of GnRH pulse generator activity, as reflected by sustained increments in plasma gonadotropin concentrations, also occurred in the absence of changes in circulating leptin levels (P>0.05). These findings indicate that the timing of the onset of puberty in male monkeys is not triggered by rising circulating leptin concentrations.

Characterization of the gene celD and its encoded product 1,4-beta-D-glucan glucohydrolase D from Pseudomonas fluorescens subsp. cellulosa.

A genomic library of Pseudomonas fluorescens subsp. cellulosa DNA constructed in pUC18 and expressed in Escherichia coli was screened for recombinants expressing 4-methylumbelliferyl beta-D-glucoside hydrolysing activity (MUGase). A single MUGase-positive clone was isolated. The MUGase hydrolysed cellobiose, cellotriose, cellotetraose, cellopentaose and cellohexaose to glucose, by sequentially cleaving glucose residues from the non-reducing end of the cello-oligosaccharides. The Km values for cellobiose and cellohexaose hydrolysis were 1.2 mM and 28 microM respectively. The enzyme exhibited no activity against soluble or insoluble cellulose, xylan and xylobiose. Thus the MUGase is classified as a 1,4-beta-D-glucan glucohydrolase (EC 3.2.1.74) and is designated 1,4-beta-D-glucan glucohydrolase D (CELD). When expressed by E. coli, CELD was located in the cell-envelope fraction; a significant proportion of the native enzyme was also associated with the cell envelope when synthesized by its endogenous host. The nucleotide sequence of the gene, celD, which encodes CELD, revealed an open reading frame of 2607 bp, encoding a protein of M(r) 92,000. The deduced primary structure of CELD was confirmed by the M(r) of CELD (85,000) expressed by E. coli and P. fluorescens subsp. cellulosa, and by the experimentally determined N-terminus of the enzyme purified from E. coli, which showed identity with residues 52-67 of the celD translated sequence. The structure of the N-terminal region of full-length CELD was similar to the signal peptides of P. fluorescens subsp. cellulosa plant-cell-wall hydrolases. Deletion of the N-terminal 47 residues of CELD solubilized MUGase activity in E. coli. CELD exhibited sequence similarity with beta-glucosidase B of Clostridium thermocellum, particularly in the vicinity of the active-site aspartate residue, but did not display structural similarity with the mature forms of cellulases and xylanases expressed by P. fluorescens subsp. cellulosa.

Characterization of hybrid proteins consisting of the catalytic domains of Clostridium and Ruminococcus endoglucanases, fused to Pseudomonas non-catalytic cellulose-binding domains.

The N-terminal 160 or 267 residues of xylanase A from Pseudomonas fluorescens subsp. cellulosa, containing a non-catalytic cellulose-binding domain (CBD), were fused to the N-terminus of the catalytic domain of endoglucanase E (EGE') from Clostridium thermocellum. A further hybrid enzyme was constructed consisting of the 347 N-terminal residues of xylanase C (XYLC) from P. fluorescens subsp. cellulosa, which also constitutes a CBD, fused to the N-terminus of endoglucanase A (EGA) from Ruminococcus albus. The three hybrid enzymes bound to insoluble cellulose, and could be eluted such that cellulose-binding capacity and catalytic activity were retained. The catalytic properties of the fusion enzymes were similar to EGE' and EGA respectively. Residues 37-347 and 34-347 of XYLC were fused to the C-terminus of EGE' and the 10 amino acids encoded by the multiple cloning sequence of pMTL22p respectively. The two hybrid proteins did not bind cellulose, although residues 39-139 of XYLC were shown previously to constitute a functional CBD. The putative role of the P. fluorescens subsp. cellulosa CBD in cellulase action is discussed.

Structural analysis by circular dichroism of some enzymes involved in plant cell wall degradation.

Three enzymes which degrade different polysaccharide components of plant cell walls have been characterized by circular dichroism (CD). A bacterial endoglucanase, which in the native state forms part of a multiprotein cellulase complex, showed a tendency to form aggregates as measured by CD. Depending on its degree of aggregation, this enzyme displayed between 50% and 100% helical structure, whereas a bacterial xylanase and a fungal polygalacturonase exhibited more beta-sheet structure. The polygalacturonase was apparently devoid of helical structure.

The non-catalytic C-terminal region of endoglucanase E from Clostridium thermocellum contains a cellulose-binding domain.

Mature endoglucanase E (EGE) from Clostridium thermocellum consists of 780 amino acid residues and has an Mr of 84,016. The N-terminal 334 amino acids comprise a functional catalytic domain. Full-length EGE bound to crystalline cellulose (Avicel) but not to xylan. Bound enzyme could be eluted with distilled water. The capacity of truncated derivatives of the enzyme to bind cellulose was investigated. EGE lacking 109 C-terminal residues (EGEd) or a derivative in which residues 367-432 of the mature form of the enzyme had been deleted (EGEb), bound to Avicel, whereas EGEa and EGEc, which lack 416 and 246 C-terminal residues respectively, did not. The specific activity of EGEa, consisting of the N-terminal 364 amino acids, was 4-fold higher than that of the full-length enzyme. The truncated derivative also exhibited lower affinity for the substrate beta-glucan than the full-length enzyme. It is concluded that EGE contains a cellulose-binding domain, located between residues 432 and 671, that is distinct from the active site. The role of this substrate-binding domain is discussed.

Xylanase B and an arabinofuranosidase from Pseudomonas fluorescens subsp. cellulosa contain identical cellulose-binding domains and are encoded by adjacent genes.

The complete nucleotide sequence of the Pseudomonas fluorescens subsp. cellulosa xynB gene, encoding an endo-beta-1,4-xylanase (xylanase B; XYLB) has been determined. The structural gene consists of an open reading frame (ORF) of 1775 bp coding for a protein of Mr 61,000. A second ORF (xynC) of 1712 bp, which starts 148 bp downstream of xynB, encodes a protein, designated xylanase C (XYLC), of Mr 59,000. XYLB hydrolyses oat spelt xylan to xylobiose and xylose, whereas XYLC releases only arabinose from the same substrate. Thus XYLB is a typical xylanase and XYLC is an arabinofuranosidase. Both enzymes bind to crystalline cellulose (Avicel), but not to xylan. The nucleotide sequences between residues 114 and 931 of xynB and xynC were identical, as were amino acid residues 39-311 of XYLB and XYLC. This conserved sequence is reiterated elsewhere in the P. fluorescens subsp. cellulosa genome. Truncated derivatives of XYLB and XYLC, in which the conserved sequence had been deleted, retained catalytic activity, but did not exhibit cellulose binding. A hybrid gene in which the 5' end of xynC, encoding residues 1-110 of XYLC, was fused to the Escherichia coli pho A' gene (encodes mature alkaline phosphatase) directed the synthesis of a fusion protein which exhibited alkaline phosphatase activity and bound to cellulose.

Endoglucanase E, produced at high level in Escherichia coli as a lacZ' fusion protein, is part of the Clostridium thermocellum cellulosome.

The Clostridium thermocellum celE gene was expressed at high level in Escherichia coli TG1 when placed under the transcriptional and translational control of lacZ' in pUC18; in the presence of a multicopy plasmid (pNM52) containing the lacIq gene, expression of full-length and truncated forms of celE was regulated by isopropyl-beta-D-thiogalactopyranoside. Catalytically active endoglucanase E (EGE) produced by E. coli was subject to proteolytic processing. The main protein species produced from full-length and truncated forms of celE was around 40 kDa in size and had an N-terminal amino acid sequence corresponding to that derived for mature EGE from the nucleotide sequence; in addition, larger species of about 75 kDa, presumably corresponding to full-size EGE, were produced by E. coli containing the full-length celE gene. Even after removal of the signal peptide sequence, EGE produced by E. coli was secreted into the periplasm. Up to 157 bp could be deleted from the 5' end of the celE gene without affecting the catalytic activity of EGE produced by E. coli. A polypeptide of Mr 86 kDa, immunoreactive with anti-EGE antiserum, was demonstrated in the high-molecular-weight, cellulose-binding multiprotein aggregate recoverable from C. thermocellum culture supernatant.

Spatial separation of protein domains is not necessary for catalytic activity or substrate binding in a xylanase.

Xylanase A (XYLA) from Pseudomonas fluorescens subspecies cellulosa shows sequence conservation with two endoglucanases from the same organism. The conserved sequence in XYLA, consisting of the N-terminal 234 residues, is not essential for catalytic activity. Full-length XYLA and a fusion enzyme, consisting of the N-terminal 100 residues of XYLA linked to mature alkaline phosphatase, bound tightly to crystalline cellulose (Avicel), but not to xylan. The capacity of truncated derivatives of the xylanase to bind polysaccharides was investigated. XYLA lacking the first 13 N-terminal amino acids did not bind to cellulose. However, a catalytically active XYLA derivative (XYLA'), in which residues 100-234 were deleted, bound tightly to Avicel. Substrate specificity, cellulose-binding capacity, specific activity and Km for xylan hydrolysis were evaluated for each of the xylanases. No differences in any of these parameters were detected for the two enzymes. It is concluded that XYLA contains a cellulose-binding domain consisting of the N-terminal 100 residues which is distinct from the active site. Spatial separation of the catalytic and cellulose-binding domains is not essential for the enzyme to function normally.

Conserved serine-rich sequences in xylanase and cellulase from Pseudomonas fluorescens subspecies cellulosa: internal signal sequence and unusual protein processing.

The complete nucleotide sequence of the xynA gene coding for a xylanase (XYLA) expressed by Pseudomonas fluorescens subspecies cellulosa, has been determined. The structural gene consists of an open reading frame of 1833 bp followed by a TAA stop codon. Confirmation of the nucleotide sequence was obtained by comparing the predicted amino acid sequence with that derived by N-terminal analysis of purified forms of the xylanase. The signal peptide present at the N terminus of mature XYLA closely resembles signal peptides of other secreted proteins. Truncated forms of the xylanase gene, in which the sequence encoding the N-terminal signal peptide had been deleted, still expressed coli. XYLA contains domains which are homologous to an endoglucanase expressed by the same organism. These structures include serine-rich sequences. Bal31 deletions of xynA revealed the extent to which these conserved sequences, in XYLA, were essential for xylanase activity. Downstream of the TAA stop codon is a G + C-rich region of dyad symmetry (delta G = 24 kcal) characteristic of E. coli Rho-independent transcription terminators.

Recombination and genome size.

Within complements the chiasma frequency per chromosome, which directly reflects the amount of recombination, is generally closely correlated with chromosome length, i.e. the chromosomal DNA content. The correlation does not apply when comparisons are made between the complements of different species. Analyses of results from three Angiosperm genera show a progressive decrease in the chiasma frequency per picogram of DNA with increase in nuclear DNA amount.