Cytocompatibility of electrospun poly-L-lactic acid membranes for Bruch's membrane regeneration using human embryonic stem cell-derived retinal pigment epithelial cells.

Cell replacement therapy is under development for dry age-related macular degeneration (AMD). A thin membrane resembling the Bruch's membrane is required to form a cell-on-membrane construct with retinal pigment epithelial (RPE) cells. These cells have been differentiated from human embryonic stem cells (hESCs) in vitro. A carrier membrane is required for cell implantation, which is biocompatible for cell growth and has dimensions and physical properties resembling the Bruch's membrane. Here a nanofiber electrospun poly-L-lactic acid (PLLA) membrane is tested for capacity to support cell growth and maturation. The requirements for laminin coating of the membrane are identified here. A porous electrospun nanofibrous PLLA membrane of ∼50 nm fiber diameter was developed as a prototype support for functional RPE cells grown as a monolayer. The need for laminin coating applied to the membrane following treatment with poly-L-ornithine (PLO), was identified in terms of cell growth and survival. Test membranes were compared in terms of hydrophilicity after laminin coating, mechanical properties of surface roughness and Young's modulus, porosity and ability to promote the attachment and proliferation of hESC-RPE cells in culture for up to 8 weeks. Over this time, RPE cell proliferation, morphology, and marker and gene expression, were monitored. The functional capacity of cell monolayers was identified in terms of transepithelial electrical resistance (TEER), phagocytosis of cells, as well as expression of the cytokines, vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF). PLLA polymer fibers are naturally hydrophobic, so their hydrophilicity was improved by pretreatment with PLO for subsequent coating with the bioactive protein laminin. They were then assessed for amount of laminin adsorbed, contact angle and uniformity of coating using scanning electron microscopy (SEM). Pretreatment with 100% PLO gave the best result over 10% PLO treatment or no treatment prior to laminin adsorption with significantly greater surface stiffness and modulus. By 6 weeks after cell plating, the coated membranes could support a mature RPE monolayer showing a dense apical microvillus structure and pigmented 3D polygonal cell morphology. After 8 weeks, PLO (100%)-Lam coated membranes exhibited the highest cell number, cell proliferation, and RPE barrier function measured as TEER. RPE cells showed the higher levels of specific surface marker and gene expression. Microphthalmia-associated transcription factor expression was highly upregulated indicating maturation of cells. Functionality of cells was indicated by expression of VEGF and PEDF genes as well as phagocytic capacity. In conclusion, electrospun PLLA membranes coated with PLO-Lam have the physical and biological properties to support the distribution and migration of hESC-RPE cells throughout the whole structure. They represent a good membrane candidate for preparation of hESC-RPE cells as a monolayer for implantation into the subretinal space of AMD patients.

The dual orexin receptor antagonist suvorexant in alcohol use disorder and comorbid insomnia: A case report.

Key Clinical Message: This case suggests using dual orexin receptor antagonists to treat alcohol use disorder and comorbid sleep disorders may be effective, commencing treatment in withdrawal and continuing it to prevent relapse. Abstract: Effective medications for the treatment of alcohol use disorder are limited. This is partially due to the heterogenous nature of the symptomatology associated with alcohol use disorder and the abundance of presenting comorbidities. One common, and often overlooked, symptom that occurs during withdrawal of alcohol use is sleep disruption. Here, we report a case study of a participant with comorbid alcohol use disorder and insomnia. This participant was treated with a dual orexin receptor antagonist, suvorexant (Belsomra®), currently approved to treat insomnia. We demonstrate improvements in alcohol cravings, physical and psychological health, and sleep outcomes with treatment. These data support abundant preclinical and emerging clinical data in this space. The findings from this case report highlight the potential for suvorexant to treat comorbid alcohol use disorder and insomnia with fully powered, randomized controlled trials moving forward.

Analysing responsible innovation along a value chain-A single-cell protein case study.

The British Standards Institution's Publicly Available Specification 440 (PAS 440) provides a Responsible Innovation Framework (RIF) that companies can use to continuously monitor the societal, environmental and health benefits and risks of their innovations, as well as relevant changes to the supply chain and regulations. PAS 440 is intended to help companies achieve the benefits of innovation in a timely manner and avoid any potential harm or unintended misuse of a new product, process or service. Here, the authors have applied the PAS 440 RIF to a novel single-cell protein (SCP) animal feed ingredient taking into consideration the perspectives of the value chain partners (VCPs), companies and laboratories involved in an Innovate UK research project. The authors' findings show how VCPs can use PAS440 to demonstrate that they are innovating responsibly. Using this approach to responsible innovation along the value chain-from manufacturing scale-up, through regulatory approval, to incorporation in animal feed and from there to food on supermarket shelves-can support the development of innovations that contribute to the economic and environmental sustainability of the animal feed sector. The authors conclude that the PAS 440 Guide can facilitate the progress of a new product throughout a value chain and contribute to coordinating responsible behaviour among companies involved in the value chain.

The effect of myeloablative radiation on urinary bladder mast cells.

Radiation-induced cystitis is an inflammatory condition affecting the urinary bladder, which can develop as a side effect of abdominopelvic radiotherapy, specifically external-beam radiation therapy or myeloablative radiotherapy. A possible involvement of mast cells in the pathophysiology of radiation-induced cystitis has been indicated in cases of external-beam radiation therapy; however, there is no evidence that these findings apply to the myeloablative aetiology. As such, this study investigated potential changes to urinary bladder mast cell prevalence when exposed to myeloablative radiation. Lethally irradiated C57BL/6J mice that received donor rescue bone marrow cells exhibited an increased mast cell frequency amongst host leukocytes 1 week following irradiation. By 4 weeks, no significant difference in either frequency or cell density was observed. However mast cell diameter was smaller, and a significant increase in mast cell number in the adventitia was observed. This study highlights that mast cells constitute a significant portion of the remaining host leukocyte population following radiation exposure, with changes to mast cell distribution and decreased cell diameter four weeks following radiation-induced injury.

Broad host range phages target global Clostridium perfringens bacterial strains and clear infection in five-strain model systems.

Clostridium perfringens is a prevalent bacterial pathogen in poultry, and due to the spread of antimicrobial resistance, alternative treatments are needed to prevent and treat infection. Bacteriophages (phages), viruses that kill bacteria, offer a viable option and can be used therapeutically to treat C. perfringens infections. The aim of this study was to isolate phages against C. perfringens strains currently circulating on farms across the world and establish their virulence and development potential using host range screening, virulence assays, and larva infection studies. We isolated 32 phages of which 19 lysed 80%-92% of our global C. perfringens poultry strain collection (n = 97). The virulence of these individual phages and 32 different phage combinations was quantified in liquid culture at multiple doses. We then developed a multi-strain C. perfringens larva infection model, to mimic an effective poultry model used by the industry. We tested the efficacy of 16/32 phage cocktails in the larva model. From this, we identified that our phage cocktail consisting of phages CPLM2, CPLM15, and CPLS41 was the most effective at reducing C. perfringens colonization in infected larvae when administered before bacterial challenge. These data suggest that phages do have significant potential to prevent and treat C. perfringens infection in poultry. IMPORTANCE: Clostridium perfringens causes foodborne illness worldwide, and 95% of human infections are linked to the consumption of contaminated meat, including chicken products. In poultry, C. perfringens infection causes necrotic enteritis, and associated mortality rates can be up to 50%. However, treating infections is difficult as the bacterium is becoming antibiotic-resistant. Furthermore, the poultry industry is striving toward reduced antibiotic usage. Bacteriophages (phages) offer a promising alternative, and to progress this approach, robust suitable phages and laboratory models that mimic C. perfringens infections in poultry are required. In our study, we isolated phages targeting C. perfringens and found that many lyse C. perfringens strains isolated from chickens worldwide. Consistent with other published studies, in the model systems we assayed here, when some phages were combined as cocktails, the infection was cleared most effectively compared to individual phage use.

First observation of a skate egg case nursery in the Ross Sea.

Areas of importance to Southern Ocean skates are poorly defined. Here, we identify a deepwater skate egg case nursery in a discrete location at ~460 m depth off Cape Adare in the Southern Ocean. This is the first confirmed observation of a skate nursery area in the Ross Sea and only the second observation for the Southern Ocean. The morphology and size of the egg cases were consistent with the genus Bathyraja and most likely belong to the Bathyraja sp. (cf. eatonii). The nursery occurs within the "no take" General Protection Zone of the Ross Sea region marine protected area, where commercial fishing is prohibited.

Sharks checking in to the sponge hotel: First internal use of sponges of the genus Agelas and family Irciniidae by banded sand catsharks Atelomycterus fasciatus.

Trawl surveys within and surrounding two northwestern Australian marine parks revealed banded sand catsharks Atelomycterus fasciatus (family Atelomycteridae) taking refuge within large sponges of the family Irciniidae (Demospongiae: Dictyoceratida) and the genus Agelas (Demospongiae: Agelasida: Agelasidae). Five sponges contained a total of 57 A. fasciatus, comprising both sexes and both immature and mature individuals ranging from 102 to 390 mm total length (TL). In the same surveys, only five A. fasciatus were captured unassociated with sponges, suggesting that sponges are an important microhabitat for A. fasciatus and may provide a daytime refuge from predators. A southerly range extension is also reported for this species.

A bacteriophage cocktail delivered in feed significantly reduced Salmonella colonization in challenged broiler chickens.

Nontyphoidal Salmonella spp. are a leading cause of human gastrointestinal infections and are commonly transmitted via the consumption of contaminated meat. To limit the spread of Salmonella and other food-borne pathogens in the food chain, bacteriophage (phage) therapy could be used during rearing or pre-harvest stages of animal production. This study was conducted to determine if a phage cocktail delivered in feed is capable of reducing Salmonella colonization in experimentally challenged chickens and to determine the optimal phage dose. A total of 672 broilers were divided into six treatment groups T1 (no phage diet and unchallenged); T2 (phage diet 106 PFU/day); T3 (challenged group); T4 (phage diet 105 PFU/day and challenged); T5 (phage diet 106 PFU/day and challenged); and T6 (phage diet 107 PFU/day and challenged). The liquid phage cocktail was added to mash diet with ad libitum access available throughout the study. By day 42 (the concluding day of the study), no Salmonella was detected in faecal samples collected from group T4. Salmonella was isolated from a small number of pens in groups T5 (3/16) and T6 (2/16) at ∼4 × 102 CFU/g. In comparison, Salmonella was isolated from 7/16 pens in T3 at ∼3 × 104 CFU/g. Phage treatment at all three doses had a positive impact on growth performance in challenged birds with increased weight gains in comparison to challenged birds with no phage diet. We showed delivering phages via feed was effective at reducing Salmonella colonization in chickens and our study highlights phages offer a promising tool to target bacterial infections in poultry.

Can the broiler industry rely on results of existing life cycle assessment and environmental assessments studies to inform broilers' nutritional strategies?

The goal of this systematic review is to investigate the applicability of the results from existing life cycle analysis (LCA) and environmental assessments studies in informing nutritional strategies for environmentally sustainable poultry meat production. This paper reports on a Rapid Evidence Assessment (REA) of articles published between 2000 and 2020. The studies reviewed were conducted in developed countries including UK, France, Germany, Sweden, Norway, The Netherlands, Denmark, Belgium, Canada, and USA. All articles were written in English. The REA includes studies on LCA of differing strains of meat poultry and production systems, studies on poultry manure emission and studies on environmental assessments of plant-based feed ingredients. The review covered studies on soil carbon dynamics associated with plant-based ingredients. Web of Science, Scopus, and PubMed were used to obtain the 6,142 population articles. The multistage screening process resulted in 29 studies from which 15 studies included LCA while the rest 14 studies analyzed NH3 emission of broilers. All studies based on LCA were descriptive and did not include replications. Only 12 studies assessed the effect of interventions to reduce NH3 emission of broiler litter using replicated layout designs. It is concluded that the broiler industry in UK, EU, and North America cannot rely on results of existing LCA and environmental assessments studies to inform their nutritional strategy and poultry meat production due to a shortage of reliable in vivo data assessing interventions in controlled studies.

Skeletal stem/progenitor cells provide the niche for extramedullary hematopoiesis in spleen.

In bone marrow, the niche which supports hematopoiesis and nurtures hematopoietic stem cells (HSCs) contains perivascular reticular cells representing a subset of skeletal stem/progenitor cells (SSPCs). These stromal cells which provide the niche are lost or become inadequate during stress, disease or ageing, such that HSCs leave bone marrow and enter spleen and other peripheral sites to initiate extramedullary hematopoiesis and particularly myelopoiesis. Spleen also maintains niches for HSCs under steady-state conditions, evident since neonatal and adult spleen contain HSCs in low number and provide low-level hematopoiesis. In spleen, HSCs are found in the sinusoidal-rich red pulp region also in the vicinity of perivascular reticular cells. These cells resemble to some extent the known stromal elements reflecting HSC niches in bone marrow, and are investigated here for their characteristics as a subset of SSPCs. The isolation of spleen stromal subsets and the generation of cell lines which support HSCs and myelopoiesis in vitro has led to the identification of perivascular reticular cells which are unique to spleen. Analysis of gene and marker expression, as well as differentiative potential, identifies an osteoprogenitor cell type, reflective of one of several subsets of SSPCs described previously in bone, bone marrow and adipose tissue. The combined information supports a model for HSC niches in spleen involving perivascular reticular cells as SSPCs having osteogenic, stroma-forming capacity. These associate with sinusoids in red pulp to form niches for HSCs and to support the differentiation of hematopoietic progenitors during extramedullary hematopoiesis.

What came first, the shark or the egg? Discovery of a new species of deepwater shark by investigation of egg case morphology.

Apristurus ovicorrugatus, a new species of deepwater catshark, is described from northwestern Australia. Unique egg cases belonging to an unknown species of Apristurus prompted a more detailed investigation of Apristurus specimens off northwestern Australia. One specimen previously identified as A. sinensis collected off Dampier Archipelago was found gravid with a single egg case. Removal of this egg case confirmed that this species was responsible for producing the unique egg cases previously recorded. The egg cases of this species have strong T-shaped longitudinal ridges on the dorsal and ventral surfaces which are unique in the genus Apristurus. The ridges most closely resemble those present in Bythaelurus canescens from South America, but are larger and always T-shaped. The holotype is closest morphologically to A. sinensis but differs in having a medium brown buccal cavity (vs. jet black), ridged egg cases (vs. smooth egg cases), fewer intestinal spiral valve turns and larger pectoral fins. The holotype is also similar, and closest on a molecular level, to A. nakayai with which it shares a unique synapomorphic character, the white shiny iris (apomorphic within the genus). A late-term embryo removed from an egg case superficially resembled the holotype except in having two parallel rows of enlarged dermal denticles on the dorsolateral predorsal surface. Recent nomenclatural changes to the genera Apristurus and Pentanchus are discussed and challenged. This study highlights the important contribution that egg case morphology has on oviparous elasmobranch taxonomy.

Investigating the significance of segmental aneuploidy findings in preimplantation embryos.

Segmental aneuploidies (SAs) are structural imbalances, namely, gains or losses, involving a chromosomal segment. Most preimplantation genetic testing platforms can detect segmental imbalances greater than 5-10 Mb, either full or mosaic; however, questions remain about clinical significance. An in-depth review was performed to determine the accuracy, frequency, and types of SAs detected in preimplantation embryos. A comprehensive search of the literature revealed an incidence of approximately 8.15% in preimplantation embryos, compared with a prevalence of 3.55% in prenatal diagnosis samples. Several studies have used rebiopsy analysis to validate the accuracy and reproducibility of such findings in blastocyst-stage embryos. A comparison of these studies yielded a mean confirmation rate of SAs slightly higher than 30%. This result could be attributed to their mitotic origin as well as to the technical limitations of preimplantation genetic testing. In addition, the few available studies in which embryos with a segmental finding were transferred in utero are analyzed to discuss the reproductive competence of such embryos. Except for 1 study, all outcomes were described for segmental embryos in a mosaic state. As a result, there is still insufficient evidence to provide accurate information about the effect of segmental imbalances on embryonic reproductive competence and to determine gestational and newborn risks.

Seamount coral reefs are egg case nurseries for deep-sea skates.

Egg case nurseries of the boreal skate (Amblyraja hyperborea) and Richardson's skate (Bathyraja richardsoni) were defined and mapped on a bathyal seascape (c. 500-1900 m depths) south of Tasmania, Australia, using 99 towed-camera transects (157 linear km; N = 50,858 images). In total, 738 skate egg cases were observed (present in 240 images, absent in 50,618); among 113 egg cases examined to identify parent species, 70% were A. hyperborea, 10% B. richardsoni and 20% unidentified Bathyraja species. "Recently laid" egg cases were differentiated from "aged" ones by classifying their colour and condition. The great majority (98%) of egg cases were observed in c. 1100-1400 m depths on seamounts (15 of 36 surveyed), not seamount bases or adjacent continental slope. Egg cases were associated with reefs formed by accumulated skeletal matrix of the stony coral Solenosmilia variabilis, with >90% egg cases (including most of those recently laid) observed on living S. variabilis that characterises a "coral zone" in c. 950-1350 m depths. Water in the coral zone is warmer (+0.66 to 2.37°C) than at the deep distributional limits of adult A. hyperborea and B. richardsoni (2000 and 3000 m, respectively), potentially providing for accelerated embryonic development. Co-occurrence with living coral infers an energetically favourable local-scale hydrodynamic environment for egg cases, particularly on seamount peaks, where increased water flow over egg cases would avert smothering by suspended sediment, and compensate for lower oxygen concentration compared to deeper depths occupied by adult skates. Criteria identifying egg case nurseries are strongly met for A. hyperborea at Seamount Z110 (468 egg cases of varied ages, maximum density of 5.47 m-2 ) and to a lesser extent on five others (Seamounts K1, Z16, Hill U, Z5 and Hill V). An abundance (density) criterion for defining nurseries needs to be flexible because it is a spatially scale-dependent measure that differs between surveys according to the tools and design employed. Off Australia, coral reef egg case nursery habitat is restricted to a narrow depth range in temperate latitudes where it is scarce and impacted by historical bottom trawl fishing in many locations. There has been effective conservation of nursery habitat, however, because four of the six nursery sites identified here and extensive coral reef areas are protected within marine parks.

A Placebo-Controlled Randomized Trial of Vigabatrin in the Management of Acute Alcohol Withdrawal.

OBJECTIVE: To undertake a double blinded randomised placebo-controlled trial to assess the efficacy of vigabatrin, a GABA-transaminase inhibitor, as a benzodiazepine sparing agent in the management of acute alcohol withdrawal syndrome in a residential setting. METHODS: We enrolled 120 patients with alcohol use disorder who were randomly assigned to either treatment with vigabatrin (2g/day for 4 days) or placebo. The primary outcome was defined as the number of participants in each treatment arm needing diazepam for withdrawal management. A secondary outcome prespecified was the total dose of diazepam received by participants in each treatment arm. Participants were recruited on admission to a residential withdrawal unit at St Vincent's Hospital Melbourne from December 2014 to April 2019. RESULTS: No significant difference was observed in the number of participants requiring benzodiazepines during their residential withdrawal stay with 44 participants (78.6%) in placebo arm requiring at least one dose of diazepam compared to 38 (66.7%) in vigabatrin arm (p = .156). An 18.1% difference was observed between the proportion of participants who received a total dose of >100mg of diazepam during their residential withdrawal stay in placebo arm (32.1%), compared to vigabatrin arm (14.0%, p = .022). There were higher rates of reported adverse events in placebo arm with nine (15.0%) participants reporting adverse events compared with two (3.3%) participants in vigabatrin arm (p = .027). CONCLUSION: Vigabatrin significantly reduced the number of participants requiring >100mg diazepam over the course of their alcohol withdrawal and was associated with a reduction in adverse effects when compared to placebo.

Genetic associations with polycystic ovary syndrome: the role of the mitochondrial genome; a systematic review and meta-analysis.

BACKGROUND: Polycystic ovary syndrome (PCOS) remains the most common female reproductive endocrine disorder. Genetic studies have predominantly focused on the role of the nuclear genome, while the contribution of mitochondrial genetics in PCOS remains largely unknown. AIM: This study aims to systematically evaluate the literature regarding the associations between the mitochondrial genome and PCOS. METHODS: A literature search focused on PCOS and mitochondrial genetics was conducted on (1) MEDLINE, (2) EMBASE and (3) The Cochrane Library (CENTRAL and Cochrane Reviews). Search results were screened for eligibility, and data involving genetic variants of mitochondrial DNA (mtDNA) were extracted. Quantitative data were presented in forest plots, and where this was not possible, data were analysed in a qualitative manner. Quality of studies was assessed using the Q-Genie tool. RESULTS: Of the 13 812 identified studies, 15 studies were eligible for inclusion, with 8 studies suitable for meta-analysis. Women with PCOS showed higher frequencies of a 9 bp deletion, and aberrant single nucleotide polymorphisms (SNPs) in the ND5, A6 and 7 transfer RNA-encoding genes. They also showed lower frequencies of two SNPs in the D-loop of the genome. Women with PCOS also exhibited significantly lowered mtDNA copy number. CONCLUSION: Women with PCOS harbour genetic variants in coding and non-coding regions of the mitochondrial genome. This may disrupt the electron transport chain and lead to oxidative stress, causing apoptosis of cells and further genetic damage. However, further studies of higher quality are required to confirm these associations. PROSPERO REGISTRATION NUMBER: CRD42021267991.

Further description of the Kerguelen sandpaper skate Bathyraja irrasa (Rajiformes: Arhynchobatidae) based on additional specimens, including egg cases and embryos.

Adult specimens, additional juvenile specimens, egg cases and embryos were used to provide a more detailed anatomical description of the Kerguelen sandpaper skate Bathyraja irrasa, a species of skate endemic to the Kerguelen Plateau and listed as Vulnerable by the IUCN Red List of Threatened Species. The morphological and meristic data reveal a relatively high level of intraspecific variation, mostly related to size. Egg cases are described for the first time and were shown to vary in colour and fouling depending on the length of time spent in the marine environment. Embryos removed from egg cases represent all stages of embryonic development in this species and include the largest embryo recorded for this species, i.e., 230 mm total length (LT ), which increases the range of size at hatching of B. irrasa to 178-230 mm LT . A number of morphometric and meristic characters varied ontogenetically in B. irrasa, in particular relative tail length, number of tail thorns in the median row and the size of orbits. This study highlights the importance of describing intraspecific variation in species and the importance of egg cases to taxonomic and biological research on oviparous species.

Age-related fertility decline: is there a role for elective ovarian tissue cryopreservation?

Age-related fertility decline (ARFD) is a prevalent concern amongst western cultures due to the increasing age of first-time motherhood. Elective oocyte and embryo cryopreservation remain the most established methods of fertility preservation, providing women the opportunity of reproductive autonomy to preserve their fertility and extend their childbearing years to prevent involuntary childlessness. Whilst ovarian cortex cryopreservation has been used to preserve reproductive potential in women for medical reasons, such as in pre- or peripubertal girls undergoing gonadotoxic chemotherapy, it has not yet been considered in the context of ARFD. As artificial reproductive technology (ART) and surgical methods of fertility preservation continue to evolve, it is a judicious time to review current evidence and consider alternative options for women wishing to delay their fertility. This article critically appraises elective oocyte cryopreservation as an option for women who use it to mitigate the risk of ARFD and introduces the prospect of elective ovarian cortex cryopreservation as an alternative.

Extramedullary hematopoiesis: mesenchymal stromal cells from spleen provide an in vitro niche for myelopoiesis.

Murine spleen has been shown to harbour stromal cells that support hematopoiesis with production of myeloid antigen-presenting cells. Similar stromal lines have now been isolated from long-term cultures (LTC) of human spleen. When human progenitor populations from spleen, bone marrow and cord blood were employed as a source of progenitors for co-culture above splenic stromal lines, myelopoiesis was supported. Human splenocytes gave production of predominantly myeloid dendritic-like cells, with minor subsets resembling conventional dendritic cells (cDC) cells, and myeloid or monocyte-derived DC. Human bone marrow progenitors gave rise to myelopoiesis from hematopoietic progenitors, while human cord blood supported limited myelopoiesis from existing myeloid precursors. Transcriptome analysis compared two stromal lines differing in myelopoietic support capacity. Gene profiling revealed both stromal lines to reflect perivascular reticular cells with osteogenic characteristics. However, the 5C6 stroma which failed to support hematopoiesis uniquely expressed several inhibitors of the WNT pathway. Combined data now show that splenic stroma of both human and murine origin provides a mesenchymal stromal cell microenvironment which is WNT pathway-dependent, and which supports in vitro myelopoiesis with production of specific subsets of myeloid and dendritic-like cells.