Experimental Infection of Mexican Free-Tailed Bats (Tadarida brasiliensis) with SARS-CoV-2.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) virus is thought to have originated in wild bats from Asia, and as the resulting pandemic continues into its third year, concerns have been raised that the virus will expand its host range and infect North American wildlife species, including bats. Mexican free-tailed bats (Tadarida brasiliensis) live in large colonies in the southern United States, often in urban areas and, as such, could be exposed to the virus from infected humans. We experimentally challenged wild T. brasiliensis with SARS-CoV-2 to determine the susceptibility, reservoir potential, and population impacts of infection in this species. Of 10 bats oronasally inoculated with SARS-CoV-2, 5 became infected and orally excreted moderate amounts of virus for up to 18 days postinoculation. These five subjects all seroconverted and cleared the virus before the end of the study with no obvious clinical signs of disease. We additionally found no evidence of viral transmission to uninoculated subjects. These results indicate that while T. brasiliensis are susceptible to SARS-CoV-2 infection, infection of wild populations of T. brasiliensis would not likely cause mortality. However, the transmission of SARS-CoV-2 from T. brasiliensis to or from humans, or to other animal species, is a possibility requiring further investigation to better define. IMPORTANCE As the COVID-19 pandemic has continued for 3+ years, there has been increasing concern that the SARS-CoV-2 virus will enter wildlife populations and potentially create new reservoirs where the virus could adapt to a new host and create variants. This is particularly possible with species that reside in man-made structures, in proximity to infected human populations. Mexican free-tailed bats (Tadarida brasiliensis) live in large colonies, often in urban settings and, thus, can be exposed by infected humans and potentially transmit the virus to new hosts. We experimentally challenged T. brasiliensis with SARS-CoV-2 and revealed that they are susceptible to the virus and excrete moderate amounts for up to 18 days postinoculation. This is important information for wildlife biologists, wildlife rehabilitation workers, and the general public that may contact these animals.

Enhanced stability of microRNA expression facilitates classification of FFPE tumour samples exhibiting near total mRNA degradation.

BACKGROUND: As degradation of formalin-fixed paraffin-embedded (FFPE) samples limits the ability to profile mRNA expression, we explored factors predicting the success of mRNA expression profiling of FFPE material and investigated an approach to overcome the limitation. METHODS: Bladder (n=140, stored 3-8 years) and cervix (n=160, stored 8-23 years) carcinoma FFPE samples were hybridised to Affymetrix Exon 1.0ST arrays. Percentage detection above background (%DABG) measured technical success. Biological signal was assessed by distinguishing cervix squamous cell carcinoma (SCC) and adenocarcinoma (AC) using a gene signature. As miR-205 had been identified as a marker of SCC, precursor mir-205 was measured by Exon array and mature miR-205 by qRT-PCR. Genome-wide microRNA (miRNA) expression (Affymetrix miRNA v2.0 arrays) was compared in eight newer FFPE samples with biological signal and eight older samples without. RESULTS: RNA quality controls (QCs) (e.g., RNA integrity (RIN) number) failed to predict profiling success, but sample age correlated with %DABG in bladder (R=-0.30, P<0.01) and cervix (R=-0.69, P<0.01). Biological signal was lost in older samples and neither a signature nor precursor mir-205 separated samples by histology. miR-205 qRT-PCR discriminated SCC from AC, validated by miRNA profiling (26-fold higher in SCC; P=1.10 × 10(-5)). Genome-wide miRNA (R=0.95) and small nucleolar RNA (R=0.97) expression correlated well in the eight newer vs older FFPE samples and better than mRNA expression (R=0.72). CONCLUSION: Sample age is the best predictor of successful mRNA profiling of FFPE material, and miRNA profiling overcomes the limitation of age and copes well with older samples.

Experimental infection of Mexican free-tailed bats ( Tadarida brasiliensis) with SARS-CoV-2.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) virus originated in wild bats from Asia, and as the resulting pandemic continues into its third year, concerns have been raised that the virus will expand its host range and infect North American wildlife species, including bats. Mexican free-tailed bats ( Tadarida brasiliensis : TABR) live in large colonies in the southern United States, often in urban areas, and as such, could be exposed to the virus from infected humans. We experimentally challenged wild TABR with SARS-CoV-2 to determine the susceptibility, reservoir potential, and population impacts of infection in this species. Of nine bats oronasally inoculated with SARS-CoV-2, five became infected and orally excreted moderate amounts of virus for up to 18 days post inoculation. These five subjects all seroconverted and cleared the virus before the end of the study with no obvious clinical signs of disease. We additionally found no evidence of viral transmission to uninoculated subjects. These results indicate that while TABR are susceptible to SARS-CoV-2 infection, infection of wild populations of TABR would not likely cause mortality. However, the transmission of SARS-CoV-2 from TABR to or from humans, or to other animal species, is a distinct possibility requiring further investigation to better define.

Exon-array profiling unlocks clinically and biologically relevant gene signatures from formalin-fixed paraffin-embedded tumour samples.

BACKGROUND: Degradation and chemical modification of RNA in formalin-fixed paraffin-embedded (FFPE) samples hamper their use in expression profiling studies. This study aimed to show that useful information can be obtained by Exon-array profiling archival FFPE tumour samples. METHODS: Nineteen cervical squamous cell carcinoma (SCC) and 9 adenocarcinoma (AC) FFPE samples (10-16-year-old) were profiled using Affymetrix Exon arrays. The gene signature derived was tested on a fresh-frozen non-small cell lung cancer (NSCLC) series. Exploration of biological networks involved gene set enrichment analysis (GSEA). Differential gene expression was confirmed using Quantigene, a multiplex bead-based alternative to qRT-PCR. RESULTS: In all, 1062 genes were higher in SCC vs AC, and 155 genes higher in AC. The 1217-gene signature correctly separated 58 NSCLC into SCC and AC. A gene network centered on hepatic nuclear factor and GATA6 was identified in AC, suggesting a role in glandular cell differentiation of the cervix. Quantigene analysis of the top 26 differentially expressed genes correctly partitioned cervix samples as SCC or AC. CONCLUSION: FFPE samples can be profiled using Exon arrays to derive gene expression signatures that are sufficiently robust to be applied to independent data sets, identify novel biology and design assays for independent platform validation.

Maternal alcohol and neonatal health.

The disappearance of alcohol from blood following a single dose is a function of age, but by the time the animals are 90 days old the results are consistent from animal to animal. A single dose given to a pregnant female is distributed between fetus and maternal tissues and fluids, with a portion of the alcohol trapped in the amniotic fluid. Kittens born to mothers receiving alcohol during the last 2 weeks of pregnancy demonstrate hyperactivity, small size with unimpaired growth rate, and slow maturation of the righting reflex. It would require more experimentation to decide whether these changes are due to alcohol withdrawal or to toxic effects of the alcohol per se.

Managing Medicaid managed care: are states becoming prudent purchasers?

This paper examines the extent to which five states are becoming "prudent purchasers" in their oversight of Medicaid managed care. Our conclusions are mixed. These states are making more sustained efforts along these lines than most private purchasers are and have improved the amount and quality of the data they collect on the experiences of Medicaid clients when compared with the traditional fee-for-service program. They have been less successful in ensuring data quality that is adequate to support contracting decisions and in developing the analytical or political capacity to use data to "manage" the managed care system. Becoming a prudent purchaser appears to be a complex task for states that may prove difficult to achieve.

The effect of propranolol on blood pressure and central MOPEG concentration in the conscious spontaneously hypertensive rat.

The total concentration of the norepinephrine metabolite 3-methoxy-4-hydroxyphenylethyl glycol (MOPEG) and propranolol concentration was measured in the medulla, pons, hypothalamus and thoracic spinal cord of conscious spontaneously hypertensive rats during the development of the antihypertensive effect of a single dose of d,l-propranolol (10 mg/kg s.c.). Propranolol significantly increased MOPEG concentration by 21% in the thoracic spinal cord (2 h) and 16% in the hypothalamus (4 h), but no consistent pattern of change in regional MOPEG concentration was found in relation to the time course of the antihypertensive effect of propranolol.

Tissue distribution and antitumor activity of topotecan delivered by intracerebral clysis in a rat glioma model.

OBJECTIVE: Intracerebral clysis is a drug delivery technique that depends on convection-enhanced microinfusion to achieve therapeutic drug levels within the brain. In this study, brain tumor-bearing rats were treated with topotecan delivered systemically and by the intracerebral clysis method. Our objective was to determine the efficacy and tissue distribution of topotecan delivered by intracerebral clysis. METHODS: The C6/Wistar rat glioma model was used after a thymidine incorporation assay determined topotecan sensitivity of C6 cells in vitro. Long-term survival of animals provided objective measurements of efficacy; records of animal weight during treatment and neurological status served to approximate toxicity. Topotecan tissue penetration was measured in samples of ex vivo tumor and surrounding brain tissue with high-pressure liquid chromatography. RESULTS: Dose escalation demonstrated significant sensitivity of C6 glioma cells to topotecan (median lethal dose, 0.19 micromol/L). Eleven of 12 rats bearing established intracerebral C6 glioma and receiving topotecan by intracerebral clysis survived beyond the end point of 120 days; no untreated control or systemically treated animal survived beyond 26 days (n = 18; P < 0.005). Histopathological assessment of animals demonstrated significant tumor masses in the brains of intraperitoneally treated animals and untreated control animals. In contrast, no residual tumor was found in the brains of intracerebral clysis groups. Animal weights during treatment were markedly reduced by intraperitoneal dosing (n = 6) but not by low-dose intracerebral clysis (32 microg/kg/d for 5 d; n = 6). None of the low-dose intracerebral clysis-treated animals demonstrated neurological toxicity, and one high-dose intracerebral clysis-treated animal (160 microg/kg/d for 2 d; n = 6) died during follow-up. Topotecan was detected well beyond the boundaries of the tumor and even in the contralateral hemisphere in animals treated with intracerebral clysis. CONCLUSION: Topotecan delivered by the intracerebral clysis method is effective for treatment of brain tumors in the rat glioma model. These studies provide compelling justification for further preclinical testing to formally evaluate toxicity and efficacy with variable dosing schedules.

Limitations of the C6/Wistar rat intracerebral glioma model: implications for evaluating immunotherapy.

OBJECTIVE: Intracranial rat glioma models are a useful method for evaluating the efficacy and toxicity of novel therapies for malignant glioma. The C6/Wistar model has been used extensively as a reproducible in vivo model for studying primary brain tumors including anti-glioma immune responses. The objective of the present study is to provide in vivo evidence that the C6 rat glioma model is allogeneic within Wistar rats and is therefore inappropriate for evaluating immune responses. METHODS: Growth patterns and immune responses of C6 cells implanted into the brain and flank of Wistar rats were analyzed and compared to an immunogenic syngeneic model (9L/Fischer). RESULTS: Wistar rats with C6 tumors developed a potent humoral and cellular immune response to the tumor. Wistar rats given simultaneous flank and intracerebral tumors had a survival rate of 100% compared to an 11% survival rate in control animals receiving only intracranial C6 cells. CONCLUSION: The C6 rat glioma induces a vigorous immune reaction that may mimic a specific anti-tumor response in Wistar rats. Efficacy of immunotherapy within this model must be cautiously interpreted.

Intracerebral clysis in a rat glioma model.

OBJECTIVE: Intracerebral clysis (ICC) is a new term we use to describe convection-enhanced microinfusion into the brain. This study establishes baseline parameters for preclinical, in vivo, drug investigations using ICC in a rat glioma model. METHODS: Intracranial pressure was measured, with an intraparenchymal fiber-optic catheter, in male Fischer rats 10, 15, 20, and 25 days after implantation of C6 glioma cells in the right frontal lobe (n = 80) and in control rats without tumor (n = 20), before and during ICC. A 25% albumin solution (100 microl) was infused through an intratumoral catheter at 0.5, 1.0, 2.0, 3.0, and 4.0 microl/min. Infusate distribution was assessed by infusion of fluorescein isothiocyanate-dextran (Mr 20,000), using the aforementioned parameters (n = 36). Brains were sectioned and photographed under ultraviolet light, and distribution was calculated by computer analysis (NIH Image for Macintosh). Safe effective drug distribution was demonstrated by measuring tumor sizes and apoptosis in animals treated with N,N'-bis(2-chloroethyl)-N-nitrosourea via ICC, compared with untreated controls. Magnetic resonance imaging noninvasively confirmed tumor growth before treatment. RESULTS: Intracranial pressure increased with tumor progression, from 5.5 mm Hg at baseline to 12.95 mm Hg on Day 25 after tumor cell implantation. Intracranial pressure during ICC ranged from 5 to 21 mm Hg and was correlated with increasing infusion volumes and increasing rates of infusion. No toxicity was observed, except at the higher ends of the tumor size and volume ranges. Fluorescein isothiocyanate-dextran distribution was greater with larger infusion volumes (30 microl versus 10 microl, n = 8, P < 0.05). No significant differences in distribution were observed when different infusion rates were compared while the volume was kept constant. At tolerated flow rates, the volumes of distribution were sufficient to promote adequate drug delivery to tumors. N,N'-Bis(2-chloroethyl)-N-nitrosourea treatment resulted in significant decreases in tumor size, compared with untreated controls. CONCLUSION: The C6 glioma model can be easily modified to study aspects of interstitial delivery via ICC and the application of ICC to the screening of potential antitumor agents for safety and efficacy.

Phylogenetic relationships within the family potyviridae: wheat streak mosaic virus and brome streak mosaic virus are not members of the genus rymovirus.

ABSTRACT The complete nucleotide sequence of wheat streak mosaic virus (WSMV) has been determined based on complementary DNA clones derived from the 9,384-nucleotide (nt) RNA of the virus. The genome of WSMV has a 130-nt 5' leader and 149-nt 3'-untranslated region and is polyadenylated at the 3' end. WSMV RNA encodes a single polyprotein of 3,035 amino acid residues and has a deduced genome organization typical for a member of the family Potyviridae (5'-P1/HC-Pro/P3/6K1/CI/6K2/VPg-NIa/NIb/CP-3'). Because WSMV shares with ryegrass mosaic virus (RGMV) the biological property of transmission by eriophyid mites, WSMV has been assigned to the genus Rymovirus, of which RGMV is the type species. Phylogenetic analyses were conducted with complete polyprotein or NIb protein sequences of 11 members of the family Potyviridae, including viruses of monocots or dicots and viruses transmitted by aphids, whiteflies, and mites. WSMV and the monocot-infecting, mite-transmitted brome streak mosaic virus (BrSMV) are sister taxa and share a most recent common ancestor with the whitefly-transmitted sweet potato mild mottle virus, the type species of the proposed genus "Ipomovirus." In contrast, RGMV shares a most recent common ancestor with aphid-transmitted species of the genus Potyvirus. These results indicate that WSMV and BrSMV should be classified within a new genus of the family Potyviridae and should not be considered species of the genus Rymovirus.

Prophylactic antibiotic in elective gynaecological and obstetrical major surgery.

A prospective controlled study of 580 patients who underwent non-radical major gynaecological or obstetrical surgery examined the use of prophylactic antibiotics. The test group of patients (290) showed a significantly lower (P=0.01) incidence of the total complications and the non-infective complication rate as against those among controls (290). The duration of hospitalisation was significantly reduced for both vaginal and abdominal surgery in the test group of patients as against those in the controls. There was no evidence of increased incidence of bacterial resistance or superinfection. The authors recommend the use of prophylactic antibiotic on a short term basis (for a total duration of 4 days), commencing preoperatively to achieve a significant reduction in the infective postoperative morbidity and a shorter stay by the patients.

Fibrosarcoma of the vulva: case reports and discussion.

Three cases of fibrosarcoma of the vulva are reported. In two, surgical resection resulted in remission lasting over four years. In the third case, presenting with metastasis of the lung and liver, the patient died without surgery having been carried out. The low mitotic index found in the first two cases seems to lead to a favourable prognosis, whereas the inferior vulvar localization found in the last case seems to be more dangerous because of its diffusion into the paravaginal space.

Glanzmann's thrombasthenia and pregnancy.

Glanzmann's thrombasthenia is a rare congenital disorder of platelet function manifesting as defective primary haemostasis. Bleeding episodes often require platelet transfusions, and allo-immunization to donor platelets may occur. The problems of ensuring adequate haemostatic potential for delivery of an allo-immunized pregnant female with Glanzmann's thrombasthenia are presented.

Maternal pre-pregnancy weight and placental weight determine birth weight in normal Jamaican infants.

Birth weight is related to neonatal health and long-term risk of chronic disease. Since animal studies have shown that birth outcome is related to placental function, the present project was designed to explore the relationship between birth weight and placental growth and composition with maternal factors during pregnancy among normal term pregnancies in 51 primiparous and 40 multiparous women delivering at the University Hospital of the West Indies. Both groups were followed from 15 weeks of gestation to term. The primiparous group was generally younger than the multiparous (mean age 22 +/- 4 versus 31 +/- 5 yr). They were significantly lighter (55 +/- 8 versus 61 +/- 9 kg) with a lower body mass index (21 +/- 3 versus 23 +/- 4 kg/m2) during early pregnancy, but gained more weight during pregnancy, 11 kg compared with 8 kg, respectively. The duration of pregnancy was similar for both groups. Although the size of the placenta was not significantly different between the two groups, the mean weight of the multiparous placentae was more than that of the primiparous placentae. Also, for all mothers both placental weight and initial maternal weight related directly to birth weight. Placental non collagen protein (NCP), sodium and potassium contents were significantly higher for multiparous women and were related to birth weight. The primiparous group had babies who were significantly lighter, 3.03 kg compared with 3.36 kg, for the multiparous and this could be attributed to differences in placental function and maternal weight. When account was taken of the difference in maternal weight at the start of pregnancy and the difference in placental weight, parity no longer explained any of the differences in birth weight. It is concluded that maternal body weight at the time of becoming pregnant and the early development of the placenta determine the efficiency with which nutrients might be delivered to the foetus and hence foetal growth. The difference in birth weight between primiparous and multiparous women can be explained by the differences in maternal weight at the time of becoming pregnant.

The prognostic significance of the biomarker p16 in oropharyngeal squamous cell carcinoma.

AIMS: There is an increasing incidence of human papillomavirus (HPV)-positive oropharyngeal squamous cell cancers (OPSCC) mostly associated with favourable outcomes. p16 immunohistochemistry is a surrogate marker for HPV positivity in OPSCC. The prognostic strength of p16 over traditional prognostic factors is not fully characterised. In this study, we evaluated the clinical and demographic differences between p16-positive and -negative OPSCC and characterised its prognostic strength versus traditional prognostic factors. MATERIALS AND METHODS: Formalin-fixed, paraffin-embedded blocks and clinical information from 217 OPSCC patients, treated with radiotherapy (alone or in combination with other therapies) between 2000 and 2010 were collected retrospectively. Immunohistochemistry for p16 protein was carried out; cancer-specific survival (CSS), recurrence-free survival (RFS) and locoregional control (LRC) were calculated for both univariate and multivariate analyses. RESULTS: Ninety-two per cent of the OPSCC originated from tonsil and tongue base sites, 61% were p16 positive. Patients with p16-positive OPSCC were younger (P < 0.0001), with lower alcohol (P = 0.0002) and tobacco (P = 0.0001) exposure. The tumours were less differentiated (P = 0.0069), had a lower T stage (P = 0.0027), higher nodal status (P = 0.014) and higher American Joint Committee on Cancer (AJCC) prognostic group (P = 0.0036). AJCC prognostic group was significant for RFS (P = 0.0096) and CSS (P = 0.018) in patients with p16-negative OPSCC, but not those with p16-positive tumours (P = 0.30 and 0.54). Other significant factors for CSS and RFS in univariate analysis were: pretreatment haemoglobin (P < 0.0001 and <0.0001), chemoradiotherapy (P = 0.005 and 0.03) and P16 status (P < 0.0001 and 0.0001). In multivariate analysis, p16 positivity was the strongest independent prognostic variable for both CSS, RFS and LRC (P < 0.0001, hazard ratio 4.15; 95% confidence interval 2.43-7.08), (P < 0.0001, hazard ratio 6.15; 95% confidence interval 3.57-10.61) and (P = 0.001, hazard ratio 3.74; confidence interval 1.76-7.95). CONCLUSION: This study shows that p16 is the single most important prognostic variable in OPSCC, surpassing traditional prognostic factors for both CSS and RFS. Furthermore, disease stage has no prognostic significance in p16-positive patients, highlighting the need for routine p16 assessment in OPSCC.