Nodular lesions of the buttock for 20 years: the challenge of chromoblastomycosis in non-endemic settings.

Chromoblastomycosis is an implantation mycosis of the skin caused by certain species of melanised fungi. A man in his 50s, born in Kerala but living in England for 14 years, presented with a nodular lesion on his left buttock, which had been present for 20 years. Biopsy revealed muriform cells and fungal culture isolated Fonsecaea spp, consistent with a diagnosis of chromoblastomycosis. Treatment with oral terbinafine was initiated and changed to itraconazole based on results of antifungal susceptibility. Drug intolerance and low drug levels of itraconazole necessitated change to voriconazole and topical terbinafine. Despite long-term combined therapy, the lesions worsened, and the patient opted for surgical excision abroad. Recurrence was evident at surgical sites and combined therapy continues. Chromoblastomycosis is an insidious and burdensome neglected tropical disease. Within non-endemic countries, diagnosis remains challenging. A travel history and appropriate fungal investigations are vital.

Climate warming drives a temperate-zone lizard to its upper thermal limits, restricting activity, and increasing energetic costs.

Lizards are considered vulnerable to climate change because many operate near their thermal maxima. Exposure to higher temperatures could reduce activity of these animals by forcing them to shelter in thermal refugia for prolonged periods to avoid exceeding lethal limits. While rising temperatures should reduce activity in tropical species, the situation is less clear for temperate-zone species where activity can be constrained by both low and high temperatures. Here, we measure the effects of natural variation in environmental temperatures on activity in a temperate grassland lizard and show that it is operating near its upper thermal limit in summer even when sheltering in thermal refuges. As air temperatures increased above 32 °C, lizard activity declined markedly as individuals sought refuge in cool microhabitats while still incurring substantial metabolic costs. We estimate that warming over the last two decades has required these lizards to increase their energy intake up to 40% to offset metabolic losses caused by rising temperatures. Our results show that recent increases in temperature are sufficient to exceed the thermal and metabolic limits of temperate-zone grassland lizards. Extended periods of high temperatures could place natural populations of ectotherms under significantly increased environmental stress and contribute to population declines and extinction.

A new paradigm in the bioremoval of lead, nickel, and cadmium using a cocktail of biosystems: a metagenomic approach.

Lead (Pb), nickel (Ni), and cadmium (Cd) are known for its harmful effects on the environment. Microbial community related to soil plays a pivotal role in configuring several properties of the ecosystem. Thus, remediation of such heavy metals using multiple biosystems had shown excellent bioremoval potential. The current study demonstrates the integrated approach of Chrysopogon zizanioides in combination with earthworm Eisenia fetida augmented with VITMSJ3 potent strain for the uptake of metals like Pb, Ni, and Cd from the contaminated soil. For the uptake of heavy metals, Pb, Ni, and Cd with the concentrations of 50, 100, and 150 mg kg-1 were supplemented in pots with plants and earthworms. C. zizanioides was used for bioremoval due to their massive fibrous root system which can absorb heavy metals. A substantial increase of 70-80% Pb, Ni, and Cd was found for VITMSJ3 augmented setup. A total of 12 earthworms were introduced in each setup and were tested for the toxicity and damages in the various internal structures. Reduction in malondialdehyde (MDA) content was observed in the earthworms with VITMSJ3 strain proving less toxicity and damages. Metagenomic analysis of the soil associated bacterial diversity was assessed by amplifying the V3V4 region of the 16S rRNA gene and the annotations were studied. Firmicutes were found to be the predominant genus with 56.65% abundance in the bioaugmented soil R (60) proving the detoxification of metals in the bioaugmented soil. Our study proved that a synergistic effect of plant and earthworm in association with potent bacterial strain had higher uptake of Pb, Ni, and Cd. Metagenomic analysis revealed the changes in microbial abundance in the soil before and after treatment.

An experience using historical hepatitis C data to Re-Engage: Possibilities and pitfalls during the COVID-19 pandemic.

OBJECTIVES: Public Health England (PHE) aims meet the WHO target to eliminate hepatitis C as a public health concern by 2030. One aspect of this strategy is to use historical surveillance data of anti-HCV positive patients identified by PHE to re-engage with offers of PCR testing and treatment if RNA-positive. Operational Delivery Networks (ODN), who deliver Hepatitis C treatment across 22 regions in England, are responsible for enacting this initiative. This study aims to evaluate the effectiveness of using this data with regional PCR results to re-engage HCV-infected persons in the West Midlands region of England. STUDY DESIGN: A longitudinal prospective study using historical surveillance data. METHODS: A dataset of historical anti-HCV positive antibody patients provided to the ODN by PHE was cross-referenced with HCV RNA data from 01/01/1996 to 01/01/2019 from five laboratories across the West Midlands. Letters were sent to the general practitioner and to the patients who were HCV RNA positive to invite them for repeat testing and treatment to achieve cure. RESULTS: From a dataset of 4540 anti-HCV antibody results, 31.7% (n=1440) had a PCR result: 48.1% (n=693) were PCR positive for HCV RNA. 693 letters were sent to GPs with responses from 14.2% (n=99). By May 2021, only 212 patient letters were sent (due to significant interruption by the COVID-19 pandemic) and 11.3% (n=24) replied, 17 presented for PCR testing and 4 were found to be viraemic. To date, one patient has achieved cure and three have completed treatment awaiting confirmation of cure. CONCLUSION: The use of historical anti-HCV antibody results can be used to successfully re-engage people into testing and treatment for hepatitis C, albeit with modest gains.

Pulsed echinocandin therapy in azole intolerant or multiresistant chronic pulmonary aspergillosis: A retrospective review at a UK tertiary centre.

INTRODUCTION: Chronic pulmonary aspergillosis (CPA) is a fungal disease with high mortality and morbidity. Guidelines suggest treatment with azoles as first-line therapy. However, patients often develop treatment intolerance or increasingly azole resistance. OBJECTIVES: This retrospective review assesses outcomes in azole resistant or intolerant patients with CPA treated with cyclical echinocandin therapy. METHODS: We retrospectively examined records of 25 patients with CPA treated with cyclical caspofungin, 6 of whom were either azole-resistant or azole intolerant. Baseline characteristics, high-resolution computed tomography severity scores, forced expiratory volume after 1 minute (FEV1), forced vital capacity (FVC), body mass index and serology (Aspergillus fumigatus-specific IgG, Aspergillus fumigatus-specific IgE, total IgE and CRP) were assessed before and after caspofungin. RESULTS: Of the six patients, four (66%) started caspofungin due to intolerance and two (33%) due to pan-azole resistance. On treatment, there was stability in FEV1 with an overall mortality of 33% during the follow-up period with a median survival of 875.5 days (IQR 529-1024). No significant change in serology (A. fumigatus-specific IgG and CRP was seen. CONCLUSIONS: With pulsed echinocandin therapy, azole-intolerant or pan-resistant CPA patients have similar mortality rates to azole-naïve CPA patients. Pulsed echinocandin therapy may present a strategy to stabilize CPA in patients with pan resistance or intolerance to, azole therapy.

Lesson of the month 1: A rare adverse reaction between flucloxacillin and paracetamol.

Flucloxacillin, a beta-lactam antibiotic, is a commonly prescribed antibiotic for the treatment of infections caused by staphylococci and streptococci, most notably Staphylococcus aureus Paracetamol is one of the most dispensed medications by NHS England and is used for the treatment of fever and pain.1 However most doctors are unaware that concurrent use of these drugs can cause a potentially fatal drug interaction due to pyroglutamic acidosis (PGA), also known as 5-oxoprolinaemia. PGA is a rare cause of raised anion gap metabolic acidosis due to disruption of the γ-glutamyl cycle. We report the case of a patient with multiple comorbidities who developed PGA due to coadministration of paracetamol and flucloxacillin.

Real-time imaging of intracellular hydrogen peroxide in pancreatic islets.

A real-time method to measure intracellular hydrogen peroxide (H2O2) would be very impactful in characterizing rapid changes that occur in physiologic and pathophysiologic states. Current methods do not provide the sensitivity, specificity and spatiotemporal resolution needed for such experiments on intact cells. We developed the use of HyPer, a genetic indicator for H2O2 that can be expressed in the cytosol (cyto-HyPer) or the mitochondria (mito-HyPer) of live cells. INS-1 cells or islets were permeabilized and the cytosolic HyPer signal was a linear function of extracellular H2O2, allowing fluorescent cyto-HyPer signals to be converted into H2O2 concentrations. Glucose increased cytosolic H2O2, an effect that was suppressed by overexpression of catalase. Large perturbations in pH can influence the HyPer signal, but inclusion of HEPES [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid] in the perfusate prevented pH changes, but did not affect glucose-induced cyto-HyPer signals, suggesting that this effect is largely pH-independent. Using the assay, two fundamental questions were addressed. Knockdown of superoxide dismutase 2 (SOD2), the mitochondrial form of SOD, completely suppressed glucose-induced H2O2 Furthermore, glucose also induced mitochondrial superoxide and H2O2 production, which preceded the appearance of cytosolic H2O2 Therefore, glucose-induced H2O2 largely originated from mitochondria. Finally, the glucose-induced HyPer signal was less than 1/20th of that induced by toxic levels of H2O2 Overall, the use of HyPer for real-time imaging allowed resolution of acute changes in intracellular levels of H2O2 and will have great utility for islet studies involving mechanisms of H2O2-mediated signaling and oxidative stress.

Smooth Muscle-Alpha Actin Inhibits Vascular Smooth Muscle Cell Proliferation and Migration by Inhibiting Rac1 Activity.

Smooth muscle alpha-actin (SMA) is a marker for the contractile, non-proliferative phenotype of adult smooth muscle cells (SMCs). Upon arterial injury, expression of SMA and other structural proteins decreases and SMCs acquire a pro-migratory and proliferative phenotype. To what extent SMA regulates migration and proliferation of SMCs is unclear and putative signaling pathways involved remain to be elucidated. Here, we used lentiviral-mediated gene transfer and siRNA technology to manipulate expression of SMA in carotid mouse SMCs and studied effects of SMA. Overexpression of SMA results in decreased proliferation and migration and blunts serum-induced activation of the small GTPase Rac, but not RhoA. All inhibitory effects of SMA are rescued by expression of a constitutively active Rac1 mutant (V12rac1). Moreover, reduction of SMA expression by siRNA technology results in an increased activation of Rac. Taken together, this study identifies Rac1 as a downstream target for SMA to inhibit SMC proliferation and migration.

Serum amyloid A impairs the antiinflammatory properties of HDL.

HDL from healthy humans and lean mice inhibits palmitate-induced adipocyte inflammation; however, the effect of the inflammatory state on the functional properties of HDL on adipocytes is unknown. Here, we found that HDL from mice injected with AgNO3 fails to inhibit palmitate-induced inflammation and reduces cholesterol efflux from 3T3-L1 adipocytes. Moreover, HDL isolated from obese mice with moderate inflammation and humans with systemic lupus erythematosus had similar effects. Since serum amyloid A (SAA) concentrations in HDL increase with inflammation, we investigated whether elevated SAA is a causal factor in HDL dysfunction. HDL from AgNO3-injected mice lacking Saa1.1 and Saa2.1 exhibited a partial restoration of antiinflammatory and cholesterol efflux properties in adipocytes. Conversely, incorporation of SAA into HDL preparations reduced antiinflammatory properties but not to the same extent as HDL from AgNO3-injected mice. SAA-enriched HDL colocalized with cell surface-associated extracellular matrix (ECM) of adipocytes, suggesting impaired access to the plasma membrane. Enzymatic digestion of proteoglycans in the ECM restored the ability of SAA-containing HDL to inhibit palmitate-induced inflammation and cholesterol efflux. Collectively, these findings indicate that inflammation results in a loss of the antiinflammatory properties of HDL on adipocytes, which appears to partially result from the SAA component of HDL binding to cell-surface proteoglycans, thereby preventing access of HDL to the plasma membrane.

VIT-CMJ2: Endophyte of Agaricus bisporus in Production of Bioactive Compounds.

BACKGROUND: Agaricus bisporus is an edible basidiomycete fungus. Both the body and the mycelium contain compounds comprising a wide range of antimicrobial molecules, contributing in improvement of immunity and tumor-retardation. OBJECTIVES: The presence of endophytes capable of producing bioactive compounds was investigated in Agaricus bisporus. MATERIALS AND METHODS: Endophytes from Agaricus bisporus was isolated on LB agar. The obtained isolates were characterized morphologically and biochemically. Further 16S rRNA sequencing was implemented for molecular analysis of isolates. The isolate was mass produced and the bioactive compounds were extracted using ethyl acetate, chloroform and hexane. Agar well diffusion method was carried out to seek the potential of any antimicrobial activity of the crude bioactive compounds against known pathogens. GC-MS and FT-IR analysis were performed for the identification of bioactive compounds. RESULTS: VIT-CMJ2 was identified as Enterobacter sp. as revealed by 16S rRNA sequencing. Chloroform extract of VIT-CMJ2 showed a maximum zone of inhibition of 19 mm against Salmonella typhi followed by hexane and ethyl acetate extracts. The GC-MS analysis revealed the presence of several bioactive compounds having effective antimicrobial activity like butyl ester, Behenicalcohol, S , S-dioxide derivatives and some others which were later confirmed by FT-IR spectral stretches. CONCLUSIONS: The present study shows the insight on the way endophytes interact with Agaricus bisporus; thereby improving the nutritional profile.

Effects of exendin-4, a glucagon like peptide-1 receptor agonist, on neutrophil count and inflammatory cytokines in a rat model of endotoxemia.

BACKGROUND: Sepsis remains a major cause of morbidity and mortality. A variety of strategies targeting modulation of the pro-inflammatory response associated with early sepsis have been reported without clinical success. GLP-1 enhances glucose-stimulated insulin secretion. In addition, it was shown to have anti-inflammatory effects. We hypothesized that treatment with exendin-4, a GLP-1 receptor agonist, would attenuate inflammation and improve glucose control in a lipopolysaccharide (LPS) rat model of inflammation. METHODS: Two-month-old male Wistar rats were randomly assigned to one of the following four groups: 1) treatment: intraperitoneal (IP) injection of LPS 10 mg/kg followed by exendin-4, 30 µg/kg, 10 minutes later; 2) control-1: IP injection of LPS 10 mg/kg, followed by normal saline (NS); 3) control-2: IP NS injection followed by exendin-4; 4) sham: IP injection of NS followed by another NS injection. Glucose concentration, total white blood count with absolute neutrophil count, and pro- and anti-inflammatory cytokine concentrations were measured at 0, 3, 6, and 10 hours following LPS injection. RESULTS: At 3 hours, rats injected with LPS developed neutropenia, elevated pro- and anti-inflammatory cytokines, and mild hypoglycemia. Treatment with exendin-4 significantly modulated neutropenia, and decreased pro-inflammatory cytokine concentrations (IL-1α, IL-1ß, IL-6, TNFα, and IFNγ). However, exendin-4 had no effect on IL-10 concentrations. LPS injection led to mild hypoglycemia, that was not observed in rats treated with exendin-4. Sham animals exhibited no significant change from baseline in all parameters. CONCLUSION: In this LPS model of acute early phase inflammation, treatment with exendin-4 decreased pro-inflammatory cytokine concentrations without changing IL-10 blood levels and improved neutropenia. Following LPS injection, rats developed a tendency toward hypoglycemia that improved with exendin-4. Overall our data suggest that exogenous exendin-4 mediates anti-inflammatory effects early in this rat model of endotoxin-induced inflammation.

Repeated lentivirus-mediated granulocyte colony-stimulating factor administration to treat canine cyclic neutropenia.

Cyclic neutropenia occurs in humans and gray collie dogs, is characterized by recurrent neutropenia, and is treated by repeated injections of recombinant granulocyte colony-stimulating factor (rG-CSF). As dose escalation of lentivirus may be clinically necessary, we monitored the outcome of four sequential intramuscular injections of G-CSF-lentivirus (3 × 10(7) IU/kg body weight) to a normal dog and a gray collie. In the normal dog absolute neutrophil counts were significantly increased after each dose of virus, with mean levels of 27.75 ± 3.00, 31.50 ± 1.40, 35.05 ± 1.68, and 43.88 ± 2.94 × 10(3) cells/µl, respectively (p<0.001), and elevated neutrophil counts of 31.18 ± 7.81 × 10(3) cells/µl were maintained for more than 6 years with no adverse effects. A gray collie dog with a mean count of 1.94 ± 1.48 × 10(3) cells/µl received G-CSF-lentivirus and we observed sustained elevations in neutrophil levels for more than 5 months with a mean of 26.00 ± 11.00 × 10(3) cells/µl, significantly increased over the pretreatment level (p<0.001). After the second and third virus administrations mean neutrophil counts of 15.80 ± 6.14 and 11.52 ± 4.90 × 10(3) cells/µl were significantly reduced compared with cell counts after the first virus administration (p<0.001). However, after the fourth virus administration mean neutrophil counts of 15.21 ± 4.50 × 10(3) cells/µl were significantly increased compared with the previous administration (p<0.05). Throughout the nearly 3 years of virus administrations the dog gained weight, was healthy, and showed neutrophil counts significantly higher than pretreatment levels (p<0.001). These studies suggest that patients with cyclic and other neutropenias may be treated with escalating doses of G-CSF-lentivirus to obtain a desired therapeutic neutrophil count.

BB rat Gimap gene expression in sorted lymphoid T and B cells.

AIMS: The Gimap gene family has been shown to be integral to T cell survival and development. A frameshift mutation in Gimap5, one of seven members of the Gimap family, results in lymphopenia and is a prerequisite for spontaneous type 1 diabetes (T1D) in the BioBreeding (BB) rat. While not contributing to lymphopenia, the Gimap family members proximal to Gimap5, encompassed within the Iddm39 quantitative trait locus (QTL), have been implicated in T1D. We hypothesized that expression of the Gimap family members within the Iddm39 QTL, during thymocyte development as well as in peripheral T and B cells contribute to T1D. MAIN METHODS: Cell sorted subpopulations were analyzed by quantitative real time (qRT) PCR. KEY FINDINGS: Gimap4 expression was reduced in DR.(lyp/lyp) rat double negative, double positive and CD8 single positive (SP) thymocytes while expression of Gimap8, Gimap6, and Gimap7 was reduced only in CD8 SP thymocytes. Interestingly, expression of the entire Gimap gene family was reduced in DR.(lyp/lyp) rat peripheral T cells compared to non-lymphopenic, non-diabetic DR.(+/+) rats. With the exception of Gimap6, the Gimap family genes were not expressed in B cells from spleen and mesenteric lymph node (MLN). Expression of Gimap9 was only detected in hematopoietic cells of non B cell lineage such as macrophage, dendritic or NK cells. SIGNIFICANCE: These results suggest that lack of the Gimap5 protein in the DR.(lyp/lyp) congenic rat was associated with impaired expression of the entire family of Gimap genes and may regulate T cell homeostasis in the peripheral lymphoid organs.

Sustained glucagon-like peptide 1 expression from encapsulated transduced cells to treat obese diabetic rats.

Obesity and type 2 diabetes (T2D) are two prevalent chronic diseases that have become a major public health concern in industrialized countries. T2D is characterized by hyperglycemia and islet beta cell dysfunction. Glucagon-like peptide 1 (GLP-1) promotes ß cell proliferation and neogenesis and has a potent insulinotropic effect. Leptin receptor deficient male rats are obese and diabetic and provide a model of T2D. We hypothesized that their treatment by sustained expression of GLP-1 using encapsulated cells may prevent or delay diabetes onset. Vascular smooth muscle cells (VSMC) retrovirally transduced to secrete GLP-1 were seeded into TheraCyte(TM) encapsulation devices, implanted subcutaneously and rats were monitored for diabetes. Rats that received cell implants showed mean plasma GLP-1 level of 119.3 ± 10.2pM that was significantly elevated over control values of 32.4 ± 2.9pM (P<0.001). GLP-1 treated rats had mean insulin levels of 45.9 ± 2.3ng/ml that were significantly increased over control levels of 7.3±1.5ng/ml (P<0.001). In rats treated before diabetes onset elevations in blood glucose were delayed and rats treated after onset became normoglycemic and showed improved glucose tolerance tests. Untreated diabetic rats possess abnormal islet structures characterized by enlarged islets with α-cell infiltration and multifocal vacuolization. GLP-1 treatment induced normalization of islet structures including a mantle of α-cells and increased islet mass. These data suggest that encapsulated transduced cells may offer a potential long term treatment of patients.

Prolonged survival and improved glycemia in BioBreeding diabetic rats after early sustained exposure to glucagon-like peptide 1.

BACKGROUND: Type 1 diabetes (T1D) in both humans and BioBreeding (BB) rats is an autoimmune disease that results in complete destruction of islets and insulin dependency for life. Glucagon-like peptide 1 (GLP-1) promotes beta cell proliferation and neogenesis and has a potent insulinotropic effect. We hypothesized that the expression of GLP-1 before disease onset would increase islet mass, delay diabetes and prolong survival of BB rats. METHODS: Vascular smooth muscle cells retrovirally transduced to secrete GLP-1 were seeded into TheraCyte encapsulation devices, implanted subcutaneously, and rats were monitored for diabetes. RESULTS: In untreated control rats, plasma GLP-1 levels were 34.5-39.5 pmol/l, whereas, in treated rats, plasma levels were elevated, in the range 90-250.4 pmol/l. Hypoglycemia was not detected and this was anticipated from the glucose-regulated action of GLP-1. Diabetes onset (mean + or - SEM) in untreated rats occurred at 56.5 + or - 0.6 days (n = 6) and, in GLP-1-treated rats, was delayed until 76.4 + or - 3.3 days (n = 5) (p < 0.001). After disease onset, untreated control rats showed a rapid weight loss and elevated blood glucose (>650 mg/dl) and did not survive beyond 11 days. At 5 days after diabetes onset, insulin-secreting islets were absent in untreated rats. By contrast, treated rats maintained weight for up to 143 days of age and showed insulin-secreting beta cells. CONCLUSIONS: Sustained GLP-1 expression delivered by encapsulated cells before diabetes onset in BB rats showed an improved clinical outcome, suggesting the potential for treating patients using long lasting GLP-1 analogs.

Differential effects of leptin receptor mutation on male and female BBDR Gimap5-/Gimap5- spontaneously diabetic rats.

Rodents homozygous for autosomal leptin receptor gene mutations not only become obese, insulin resistant, and hyperleptinemic but also develop a dysregulated immune system. Using marker-assisted breeding to introgress the Koletsky rat leptin receptor mutant (lepr-/lepr-), we developed a novel congenic BBDR.(lepr-/lepr-) rat line to study the development of obesity and type 2 diabetes (T2D) in the BioBreeding (BB) diabetes-resistant (DR) rat. While heterozygous lepr (-/+) or homozygous (+/+) BBDR rats remained lean and metabolically normal, at 3 wk of age all BBDR.(lepr-/lepr-) rats were obese without hyperglycemia. Between 45 and 70 days of age, male but not female obese rats developed T2D. We had previously developed congenic BBDR.(Gimap5-/Gimap5-) rats, which carry an autosomal frameshift mutation in the Gimap5 gene linked to lymphopenia and spontaneous development of type 1 diabetes (T1D) without sex differences. Because the autoimmune-mediated destruction of pancreatic islet beta-cells may be affected not only by obesity but also by the absence of leptin receptor signaling, we next generated BBDR.(lepr-/lepr-,Gimap5-/Gimap5-) double congenic rats carrying the mutation for Gimap5 and T1D as well as the Lepr mutation for obesity and T2D. The hyperleptinemia rescued end-stage islets in BBDR.(lepr-/lepr-,Gimap5-/Gimap5-) congenic rats and induced an increase in islet size in both sexes, while T1D development was delayed and reduced only in females. These results demonstrate that obesity and T2D induced by introgression of the Koletsky leptin receptor mutation in the BBDR rat result in islet expansion associated with protection from T1D in female but not male BBDR.(lepr-/lepr-,Gimap5-/Gimap5-) congenic rats. BBDR.(lepr-/lepr-,Gimap5-/Gimap5-) congenic rats should prove valuable to study interactions between lack of leptin receptor signaling, obesity, and sex-specific T2D and T1D.

Treatment of diabetic rats with encapsulated islets.

Immunoprotection of islets using bioisolator systems permits introduction of allogeneic cells to diabetic patients without the need for immunosuppression. Using TheraCyte immunoisolation devices, we investigated two rat models of type 1 diabetes mellitus (T1DM), BB rats and rats made diabetic by streptozotocin (STZ) treatment. We chose to implant islets after the onset of diabetes to mimic the probable treatment of children with T1DM as they are usually diagnosed after disease onset. We encapsulated 1000 rat islets and implanted them subcutaneously (SQ) into diabetic biobreeding (BB) rats and STZ-induced diabetic rats, defined as two or more consecutive days of blood glucose>350 mg/dl. Rats were monitored for weight and blood glucose. Untreated BB rats rapidly lost weight and were euthanized at >20% weight loss that occurred between 4 and 10 days from implantation. For period of 30-40 days following islet implantation weights of treated rats remained steady or increased. Rapid weight loss occurred after surgical removal of devices that contained insulin positive islets. STZ-treated rats that received encapsulated islets showed steady weight gain for up to 130 days, whereas untreated control rats showed steady weight loss that achieved >20% at around 55 days. Although islet implants did not normalize blood glucose, treated rats were apparently healthy and groomed normally. Autologous or allogeneic islets were equally effective in providing treatment. TheraCyte devices can sustain islets, protect allogeneic cells from immune attack and provide treatment for diabetic-mediated weight loss in both BB rats and STZ-induced diabetic rats.

Assessment of genetic diversity in the critically endangered Australian corroboree frogs, Pseudophryne corroboree and Pseudophryne pengilleyi, identifies four evolutionarily significant units for conservation.

The iconic and brightly coloured Australian northern corroboree frog, Pseudophryne pengilleyi, and the southern corroboree frog, Pseudophryne corroboree are critically endangered and may be extinct in the wild within 3 years. We have assembled samples that cover the current range of both species and applied hypervariable microsatellite markers and mitochondrial DNA sequences to assess the levels and patterns of genetic variation. The four loci used in the study were highly variable, the total number of alleles observed ranged from 13 to 30 and the average number of alleles per locus was 19. Expected heterozygosity of the four microsatellite loci across all populations was high and varied between 0.830 and 0.935. Bayesian clustering analyses in STRUCTURE strongly supported four genetically distinct populations, which correspond exactly to the four main allopatric geographical regions in which the frogs are currently found. Individual analyses performed on the separate regions showed that breeding sites within these four regions could not be separated into distinct populations. Twelve mtND2 haplotypes were identified from 66 individuals from throughout the four geographical regions. A statistical parsimony network of mtDNA haplotypes shows two distinct groups, which correspond to the two species of corroboree frog, but with most of the haplotype diversity distributed in P. pengilleyi. These results demonstrate an unexpectedly high level of genetic diversity in both species. Our data have important implications for how the genetic diversity is managed in the future. The four evolutionarily significant units must be protected and maintained in captive breeding programmes for as long as it is possible to do.

Glucose-regulated insulin production from genetically engineered human non-beta cells.

In this report we describe development and characterization of four human cell lines that are able to secrete insulin and C-peptide in response to higher concentrations of glucose. These cell lines have been developed by stably and constitutively expressing human proinsulin with a furin-cleavable site, whereas expression of furin is regulated by glucose concentration. These cell lines have been cloned and, therefore, the transgene in each cell is located in an identical location of the genome leading to a uniform expression. Cloning has also allowed us to identify cell lines with more desirable properties such as higher basal insulin secretion and/or better glucose responsiveness. We have further shown that the insulin produced by these cells is biologically active and induces normoglycemia when injected in diabetic animals. Our objective in initiating these studies was to identify a cell line that could serve as a surrogate beta cell line for therapeutic intervention in type I diabetic patients.