Generation and transmission of interlineage recombinants in the SARS-CoV-2 pandemic.

We present evidence for multiple independent origins of recombinant SARS-CoV-2 viruses sampled from late 2020 and early 2021 in the United Kingdom. Their genomes carry single-nucleotide polymorphisms and deletions that are characteristic of the B.1.1.7 variant of concern but lack the full complement of lineage-defining mutations. Instead, the remainder of their genomes share contiguous genetic variation with non-B.1.1.7 viruses circulating in the same geographic area at the same time as the recombinants. In four instances, there was evidence for onward transmission of a recombinant-origin virus, including one transmission cluster of 45 sequenced cases over the course of 2 months. The inferred genomic locations of recombination breakpoints suggest that every community-transmitted recombinant virus inherited its spike region from a B.1.1.7 parental virus, consistent with a transmission advantage for B.1.1.7's set of mutations.

Genetic diversity in vector populations influences the transmission efficiency of an important plant virus.

The transmission efficiency of aphid-vectored plant viruses can differ between aphid populations. Intra-species diversity (genetic variation, endosymbionts) is a key determinant of aphid phenotype; however, the extent to which intra-species diversity contributes towards variation in virus transmission efficiency is unclear. Here, we use multiple populations of two key aphid species that vector barley yellow dwarf virus (BYDV) strain PAV (BYDV-PAV), the grain aphid (Sitobion avenae) and the bird cherry-oat aphid (Rhopalosiphum padi), and examine how diversity in vector populations influences virus transmission efficiency. We use Illumina sequencing to characterize genetic and endosymbiont variation in multiple Si. avenae and Rh. padi populations and conduct BYDV-PAV transmission experiments to identify links between intra-species diversity in the vector and virus transmission efficiency. We observe limited variation in the transmission efficiency of Si. avenae, with transmission efficiency consistently low for this species. However, for Rh. padi, we observe a range of transmission efficiencies and show that BYDV transmission efficiency is influenced by genetic diversity within the vector, identifying 542 single nucleotide polymorphisms that potentially contribute towards variable transmission efficiency in Rh. padi. Our results represent an important advancement in our understanding of the relationship between genetic diversity, vector-virus interactions, and virus transmission efficiency.

Vitamin D levels and mortality with SARS-COV-2 infection: a retrospective two-centre cohort study.

BACKGROUND: The role of vitamin D in increased mortality with SARS-COV-2 virus, namely, COVID-19, remains uncertain. We analysed all the patients who were treated as COVID-19-positive with or without a positive swab and were tested for vitamin D levels. METHODS: This was a retrospective, study involving 1226 patients swabbed for SARS-CoV-2 between the 10 February 2020 and 1 May 2020 at two hospitals of East Sussex Healthcare NHS Trust. Patients who were swab-positive for COVID-19 or treated as COVID-19-positive on clinical grounds even though swab results were negative were included in this study. We analysed the association of vitamin D levels and mortality, assessing linear and non-linear associations. RESULTS: A total of 1226 patients had SARS-CoV-2 RNA swabs in this period with age range from 1 year to 101 years. A cohort of 433 of these patients had swabs and recent vitamin D levels anytime in the previous 3 months. Mortality rates were not found to be associated with vitamin D levels (OR=1.04, 95% CI 0.96 to 1.12). CONCLUSION: Our findings suggest similar mortality risk from COVID-19 irrespective of the levels of vitamin D. Larger prospective studies will be needed to confirm these findings.

Innervation of taste buds revealed with Brainbow-labeling in mouse.

Nerve fibers that surround and innervate the taste bud were visualized with inherent fluorescence using Brainbow transgenic mice that were generated by mating the founder line L with nestin-cre mice. Multicolor fluorescence revealed perigemmal fibers as branched within the non-taste epithelium and ending in clusters of multiple rounded swellings surrounding the taste pore. Brainbow-labeling also revealed the morphology and branching pattern of single intragemmal fibers. These taste bud fibers frequently innervated both the peripheral bud, where immature gemmal cells are located, and the central bud, where mature, differentiated cells are located. The fibers typically bore preterminal and terminal swellings, growth cones with filopodia, swellings, and rounded retraction bulbs. These results establish an anatomical substrate for taste nerve fibers to contact and remodel among receptor cells at all stages of their differentiation, an interpretation that was supported by staining with GAP-43, a marker for growing fibers and growth cones.

Human papillomavirus (HPV) type 16 E7 protein bodies cause tumour regression in mice.

BACKGROUND: Human papillomaviruses (HPV) are the causative agents of cervical cancer in women, which results in over 250 000 deaths per year. Presently there are two prophylactic vaccines on the market, protecting against the two most common high-risk HPV types 16 and 18. These vaccines remain very expensive and are not generally affordable in developing countries where they are needed most. Additionally, there remains a need to treat women that are already infected with HPV, and who have high-grade lesions or cervical cancer. METHODS: In this paper, we characterize the immunogenicity of a therapeutic vaccine that targets the E7 protein of the most prevalent high-risk HPV - type 16 - the gene which has previously been shown to be effective in DNA vaccine trials in mice. The synthetic shuffled HPV-16 E7 (16E7SH) has lost its transforming properties but retains all naturally-occurring CTL epitopes. This was genetically fused to Zera®, a self-assembly domain of the maize γ-zein able to induce the accumulation of recombinant proteins into protein bodies (PBs), within the endoplasmic reticulum in a number of expression systems. RESULTS: High-level expression of the HPV 16E7SH protein fused to Zera® in plants was achieved, and the protein bodies could be easily and cost-effectively purified. Immune responses comparable to the 16E7SH DNA vaccine were demonstrated in the murine model, with the protein vaccine successfully inducing a specific humoral as well as cell mediated immune response, and mediating tumour regression. CONCLUSIONS: The fusion of 16E7SH to the Zera® peptide was found to enhance the immune responses, presumably by means of a more efficient antigen presentation via the protein bodies. Interestingly, simply mixing the free PBs and 16E7SH also enhanced immune responses, indicating an adjuvant activity for the Zera® PBs.

The Conservative Management of Choledocholithiasis With Ursodeoxycholic Acid.

Choledocholithiasis has been defined as the presence of stones within the common bile duct (CBD) with up to one-half of the cases remaining asymptomatic. We report a case of an 84-year-old frail male admitted for the treatment of pneumonia, pleural effusion, and bacteraemia with co-incidental deranged liver function tests (LFTs). Ensuing magnetic resonance cholangiopancreatography (MRCP) noted three CBD stones; however, the patient remained asymptomatic. After discussing the benefits and risks of treatment options with the gastroenterology team, the patient refused endoscopic retrograde cholangiopancreatography (ERCP) and opted for close monitoring in the community whilst taking ursodeoxycholic acid (UDCA). In the months following, his LFTs normalised, and repeat MRCP no longer showed stones. This case demonstrates that UDCA with close monitoring may be considered a non-invasive alternative treatment of CBD stones, particularly in elderly or frail patients with multiple comorbidities.

Genomic and cytogenetic analysis of the Ceratitis capitata temperature-sensitive lethal region.

Genetic sexing strains (GSS) are an important tool in support of sterile insect technique (SIT) applications against insect pests and disease vectors. The yet unknown temperature-sensitive lethal (tsl) gene and the recently identified white pupae (wp) gene have been used as selectable markers in the most successful GSS developed so far, the Ceratitis capitata (medfly) VIENNA 8 GSS. The molecular identification of the tsl gene may open the way for its use as a marker for the development of GSS in other insect pests and disease vectors of SIT importance. Prior studies have already shown that the tsl gene is located on the right arm of chromosome 5, between the wp and Zw loci (tsl genomic region). In the present study, we used genomic, transcriptomic, bioinformatic, and cytogenetic approaches to characterize and analyze this genomic region in wild-type and tsl mutant medfly strains. Our results suggested the presence of 561 genes, with 322 of them carrying SNPs and/or insertion-deletion (indel) mutations in the tsl genomic region. Furthermore, comparative transcriptomic analysis indicated the presence of 32 differentially expressed genes, and bioinformatic analysis revealed the presence of 33 orthologs with a described heat-sensitive phenotype of Drosophila melanogaster in this region. These data can be used in functional genetic studies to identify the tsl gene(s) and the causal mutation(s) responsible for the temperature-sensitive lethal phenotype in medfly, and potentially additional genes causing a similar phenotype.

The ecology of viruses in urban rodents with a focus on SARS-CoV-2.

Wild animals are naturally infected with a range of viruses, some of which may be zoonotic. During the human COVID pandemic there was also the possibility of rodents acquiring SARS-CoV-2 from people, so-called reverse zoonoses. To investigate this, we sampled rats (Rattus norvegicus) and mice (Apodemus sylvaticus) from urban environments in 2020 during the human COVID-19 pandemic. We metagenomically sequenced lung and gut tissue and faeces for viruses, PCR screened for SARS-CoV-2, and serologically surveyed for anti-SARS-CoV-2 Spike antibodies. We describe the range of viruses that we found in these two rodent species. We found no molecular evidence of SARS-CoV-2 infection, though in rats we found lung antibody responses and evidence of neutralization ability that are consistent with rats being exposed to SARS-CoV-2 and/or exposed to other viruses that result in cross-reactive antibodies.

Vanishing Bile Duct Syndrome Associated With Non-Hodgkin's Lymphoma and Hepatitis E Virus Infection.

The vanishing bile duct syndrome (VBDS) is a condition secondary to inciting triggers resulting in destruction and eventual disappearance of intrahepatic bile ducts leading to cholestasis. The overall outcome varies and often depends on the nature of the precipitating cause. VBDS has been found to be associated with adverse drug reactions, infectious diseases, autoimmune diseases, ischemia, and humoral factors associated with malignancies and is often irreversible. The objective of this clinical case report is to highlight the need for a broad differential to include VBDS in similar scenarios to aid rapid diagnosis and management. We hope this could lead to a more favourable outcome for patients presenting with VBDS such as the one described in this case report with concurrent non-Hodgkin's lymphoma and infection with hepatitis E virus. To the best of our knowledge, this is the first ever reported case of VBDS associated with non-Hodgkin's lymphoma and hepatitis E virus infection.

COVID-19 Vaccination-Induced Rash: Does the Choice of Vaccine Matter?

With the introduction of large-scale vaccination programmes against the coronavirus disease 2019 (COVID-19), the world has now begun to visualise a possible end to the ongoing pandemic. As with any vaccination programme, reports of side effects have begun to emerge in the wake of vaccinations. Initial reports were about mild side effects, such as local inflammation, pain, and fever. However, as a significant number of the population began to receive various COVID-19 vaccines, reports of various other moderate to severe side effects have now started to emerge. Although these side effects seem to be rare, the symptoms can be severe, and information and guidelines on how to manage them are scarce. In this case series, we discuss the incidence of widespread rashes that develop in some individuals after receiving COVID-19 vaccines by both AstraZeneca (AstraZeneca plc, Cambridge, UK) and Pfizer-BioNTech (Pfizer Inc., Brooklyn, NY; BioNTech SE, Mainz, Germany). The systemic skin reaction varied from maculopapular rashes to papules and patches that were widespread and not simply localised to the vaccine injection site. Further clinical information, awareness, and guidelines for practicing clinicians need to be exigently provided as vaccination programmes approach completion and the incidences of moderate to severe side effects of COVID-19 vaccination are becoming more apparent and pervasive.

Colovesical Fistulae: The Varying Aetiologies.

The most common presenting symptoms of colovesical fistulae (CVF) are pneumaturia and fecaluria. The most important aspect remains not only to investigate the aetiology, and the degree of both severity and complexity, but also the subsequent influence of this on overall management. In a younger population, management usually consists of curative surgery. However, this may not be possible in older patients where surgical candidacy is a genuine concern and a clinical challenge arises relating to pursuing a conservative strategy. We attempted to briefly outline how two patients were managed with a similar non-surgical approach due to frailty. These cases attempt to highlight the importance of multi-disciplinary specialty input, with a view to optimising patient care.

Mesenteric ischaemia following posterior myocardial infarction.

An 84-year-old man was admitted with urinary tract infection and chest discomfort. He initially responded to conservative acute coronary syndrome management and antibiotics.On day 6 of admission, he developed acute severe abdominal pain; 12-lead electrocardiography showed widespread ST-segment depression in the anterior chest leads with ST-elevation in the posterior leads (V7-9) suggestive of an acute posterior myocardial infarction. Arterial blood gases showed severe metabolic acidosis with a lactate of 11 mmol/L.An urgent computed tomography angiography suggested acute small bowel ischaemia. The case was discussed with the on-call surgical team, who advised that, due to severe frailty, he was not fit for surgical intervention and should be managed conservatively. He was managed with intravenous heparin infusion and supportive measures, but sadly continued to deteriorate and was palliated. He died shortly afterwards.

Delayed Spontaneous Regression of Metastatic Gastric Cancer: A Case Report of a Rare Finding.

We discuss the case of a 74-year-old male who was referred with episodes of vomiting, nausea, and weight loss. Ultrasound abdomen had suggested multiple liver metastases and a triple-phase CT scan of abdomen-pelvis confirmed the same. His oesophago-gastro-duodenoscopy (OGD), also known as upper endoscopy (EGD), showed a large ulcerated lesion at the lesser curvature, which was later confirmed to be poorly differentiated adenocarcinoma on biopsy. The patient was started on palliative chemotherapy, which he tolerated poorly, but a CT scan had suggested a minimal reduction in the size of liver metastasis. He was given two cycles of chemotherapy; however, due to poor tolerance and unresponsiveness to chemotherapy, he was referred to palliative care.  The patient declined any medical support for the next six years, after which he visited his general practitioner (GP) for a follow-up review. Routine blood tests showed new-onset mild iron deficiency anaemia. He denied any symptoms. He was referred to Gastroenterology for repeat OGD, and it showed a tiny nodular area in the stomach at the site of previous cancer, which was reported as non-specific chronic inflammation on biopsy, and CT abdomen showed a marked reduction in size and number of liver metastases. On further clinical review, he reported feeling well and his anaemia resolved without any intervention.

White pupae phenotype of tephritids is caused by parallel mutations of a MFS transporter.

Mass releases of sterilized male insects, in the frame of sterile insect technique programs, have helped suppress insect pest populations since the 1950s. In the major horticultural pests Bactrocera dorsalis, Ceratitis capitata, and Zeugodacus cucurbitae, a key phenotype white pupae (wp) has been used for decades to selectively remove females before releases, yet the gene responsible remained unknown. Here, we use classical and modern genetic approaches to identify and functionally characterize causal wp- mutations in these distantly related fruit fly species. We find that the wp phenotype is produced by parallel mutations in a single, conserved gene. CRISPR/Cas9-mediated knockout of the wp gene leads to the rapid generation of white pupae strains in C. capitata and B. tryoni. The conserved phenotype and independent nature of wp- mutations suggest this technique can provide a generic approach to produce sexing strains in other major medical and agricultural insect pests.

A Retrospective Observational Study: Is Absolute Lymphocyte Count a Prognostic Marker in COVID-19?

Aim Our study aimed to find a correlation between low absolute lymphocyte count and COVID-19-related mortality. Methods This study followed a retrospective observational cohort design to analyze the data of patients who presented with symptoms and signs of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), at the Conquest Hospital and Eastbourne District General Hospital in East Sussex, United Kingdom, between February 10, 2020 and May 1, 2020, retrospectively. Survival and mortality for the first 30 days and comorbidities were analyzed for all patients who were tested for COVID-19 irrespective of swab results and had blood lymphocyte levels taken at the time of their visit to the ED and their data were analyzed for statistical significance. Results A total of 1226 patients had SARS-CoV-2 RNA identification swabs taken between February 10, 2020 and May 1, 2020. A cohort of 742 patients of these patients tested for COVID-19 also had blood lymphocyte levels measured. Overall, the lymphocyte count did not differ significantly between patients suspected to have COVID-19 infection with either positive or negative COVID-19 swab results. The lymphocyte count, however, was significantly lower in those who died from COVID-19 (p < 0.001) but when comorbidities were analyzed, we found an association between an increased number of comorbidities and a significantly decreased lymphocyte count. Conclusion Once adjusted for comorbidities, the lymphocyte count had no association with COVID-19 infection and mortality.

Exuberant neuronal convergence onto reduced taste bud targets with preservation of neural specificity in mice overexpressing neurotrophin in the tongue epithelium.

A mouse fungiform taste bud is innervated by only four to five geniculate ganglion neurons; their peripheral fibers do not branch to other buds. We examined whether the degree or specificity of this exclusive innervation pattern is influenced by brain-derived neurotrophic factor (BDNF), a prominent lingual neurotrophin implicated in taste receptoneural development. Labeled ganglion cells were counted after injecting single buds with different color markers in BDNF-lingual-overexpressing (OE) mice. To evaluate the end-organs, taste buds and a class of putative taste receptor cells were counted from progeny of BDNF-OE mice crossbred with green fluorescent protein (GFP) (gustducin) transgenic mice. Fungiform bud numbers in BDNF-OE mice are 35%, yet geniculate neuron numbers are 195%, of wild-type mice. Neurons labeled by single-bud injections in BDNF-OE animals were increased fourfold versus controls. Injecting three buds, each with different color markers, resulted in predominantly single-labeled ganglion cells, a discrete innervation pattern similar to controls. Thus, hyper-innervation of BDNF-OE buds involves many neurons innervating single buds, not increased fiber branching. Therefore, both wild-type and BDNF-OE mice exhibit, in fungiform buds, the same, "discrete" receptoneural pattern, this despite dramatic neurotrophin overexpression-related decreases in bud numbers and increases in innervation density. Hyperinnervation did not affect GFP positive cell numbers; proportions of GFP cells in BDNF-OE buds were the same as in wild-type mice. Total numbers of ganglion cells innervating buds in transgenic mice are similar to controls; the density of taste input to the brain appears maintained despite dramatically reduced receptor organs and increased ganglion cells.

The influence of the extensional viscosity of very low concentrations of high molecular mass water-soluble polymers on atomisation and droplet impact.

BACKGROUND: Water-soluble polymers are increasingly added to herbicide and pesticide formulations at very low concentrations (100-1000 mg L(-1)) in order to control the spray characteristics, notably to reduce spray drift and influence droplet bounce. The incorporation of polymeric adjuvants improves the efficacy of the spray solutions, thus enabling crop growers to maximise the performance of agrochemical sprays at lower dose rates of active ingredient. It is important to establish a fundamental understanding of how polymers influence the processes involved in droplet deposition. RESULTS: The shear and extensional viscosities of a series of high molecular mass (M(w)) poly(acrylamides) (M(w) approximately 10(6)-10(7)) have been determined at very low concentrations (100-1000 mg L(-1)). The polymer solutions demonstrated typical shear thinning characteristics under shear, and strain hardening behaviour under extension above a critical strain rate. The presence of the polymers was shown to increase the size of droplets produced in atomisation using an agricultural spray nozzle, as measured by laser diffraction. This was attributed to the increase in the extensional viscosity at the strain rates generated under pressure in the spray nozzle and was a function of both polymer concentration and M(w). In addition, the presence of polymer was found to have a significant influence on droplet bounce. CONCLUSIONS: The presence of very low concentrations of high molecular mass poly(acrylamides) significantly influences the size of droplets formed on atomisation and subsequent bounce characteristics. Large extensional viscosities generated above a critical strain rate are responsible for both processes.

Evaluation of methods to reduce formaldehyde levels of cadavers in the dissection laboratory.

Dissection of conventionally embalmed cadavers exposes students, staff, and faculty to formaldehyde, a probable carcinogen. Therefore, prudent practices should seek to minimize formaldehyde exposure. In this study, we evaluated two commercially available chemicals, InfuTrace and Perfect Solution, for their effectiveness in reducing ambient formaldehyde levels. Four cadavers embalmed conventionally with formaldehyde and/or with the above agents were compared for their formaldehyde levels under conditions that strictly controlled for air circulation and for locations and methods of testing, and during activities that simulated student dissecting. For InfuTrace, one cadaver was reinfused with InfuTrace after initial standard perfusion with formaldehyde; a second cadaver had InfuTrace injected into the thoracic and abdominal body cavities after formaldehyde perfusion. For Perfect Solution, the product was used for embalming a third cadaver in lieu of formaldehyde. For a control, a fourth cadaver was embalmed with the standard formaldehyde solution. Testing of personal and ambient room air samples and of fluid obtained from the cadavers was performed and analyzed in a blinded fashion. Results indicated that both Perfect Solution, substituted for standard formaldehyde embalming, and InfuTrace infused through the vasculature after formaldehyde embalming, resulted in lower concentrations of formaldehyde than embalming with formaldehyde solution alone or in combination with body cavity injection of InfuTrace. These differences in formaldehyde concentrations are consistent across measuring methods, for example, of room air, of breathing zone air during cadaver handling and dissection, and of liquid samples obtained from the cadavers. Perfect Solution yielded suboptimum fixation and a different texture, color, and smell than the formaldehyde treatments.

Optimizing a Human Papillomavirus Type 16 L1-Based Chimaeric Gene for Expression in Plants.

Human papillomaviruses (HPVs) are the causative agents of cervical cancer, the fourth most prevalent cancer in women worldwide. The major capsid protein L1 self-assembles into virus-like particles (VLPs), even in the absence of the minor L2 protein: such VLPs have successfully been used as prophylactic vaccines. There remains a need, however, to develop cheaper vaccines that protect against a wider range of HPV types. The use of all or parts of the L2 minor capsid protein can potentially address this issue, as it has sequence regions conserved across several HPV types, which can elicit a wider spectrum of cross-neutralizing antibodies. Production of HPV VLPs in plants is a viable option to reduce costs; the use of a L1/L2 chimera which has previously elicited a cross-protective immune response is an option to broaden cross-protection. The objective of this study was to investigate the effect of codon optimization and of increasing the G+C content of synthetic L1/L2 genes on protein expression in plants. Additionally, we replaced varying portions of the 5' region of the L1 gene with the wild type (wt) viral sequence to determine the effect of several negative regulatory elements on expression. We showed that GC-rich genes resulted in a 10-fold increase of mRNA levels and 3-fold higher accumulation of proteins. However, the highest increase of expression was achieved with a high GC-content human codon-optimized gene, which resulted in a 100-fold increase in mRNA levels and 8- to 9-fold increase in protein levels. Changing the 5' end of the L1 gene back to its wt sequence decreased mRNA and protein expression. Our results suggest that the negative elements in the 5' end of L1 are inadvertently destroyed by changing the codon usage, which enhances protein expression.

Discrete innervation of murine taste buds by peripheral taste neurons.

The peripheral taste system likely maintains a specific relationship between ganglion cells that signal a particular taste quality and taste bud cells responsive to that quality. We have explored a measure of the receptoneural relationship in the mouse. By injecting single fungiform taste buds with lipophilic retrograde neuroanatomical markers, the number of labeled geniculate ganglion cells innervating single buds on the tongue were identified. We found that three to five ganglion cells innervate a single bud. Injecting neighboring buds with different color markers showed that the buds are primarily innervated by separate populations of geniculate cells (i.e., multiply labeled ganglion cells are rare). In other words, each taste bud is innervated by a population of neurons that only connects with that bud. Palate bud injections revealed a similar, relatively exclusive receptoneural relationship. Injecting buds in different regions of the tongue did not reveal a topographic representation of buds in the geniculate ganglion, despite a stereotyped patterned arrangement of fungiform buds as rows and columns on the tongue. However, ganglion cells innervating the tongue and palate were differentially concentrated in lateral and rostral regions of the ganglion, respectively. The principal finding that small groups of ganglion cells send sensory fibers that converge selectively on a single bud is a new-found measure of specific matching between the two principal cellular elements of the mouse peripheral taste system. Repetition of the experiments in the hamster showed a more divergent innervation of buds in this species. The results indicate that whatever taste quality is signaled by a murine geniculate ganglion neuron, that signal reflects the activity of cells in a single taste bud.