Development of polymorphic microsatellite markers for the microendemic pupfishes Cyprinodon julimes and C. pachycephalus

We developed microsatellite loci for the Julimes pupfish, Cyprinodon julimes. Twenty-five loci were screened across 19 individuals from Julimes Spring, Chihuahua, Mexico. The number of alleles per locus ranged from 2 to 14, observed heterozygosity ranged from 0.105 to 0.947, and the probability of identity values ranged from 0.022 to 0.588. We then tested for cross-amplification in the bighead pupfish, C. pachycephalus; twenty-three individuals from San Diego de Alcalá, Chihuahua, Mexico, were screened across the 20 loci that amplified cleanly. These new loci will be used for long-term genetic monitoring of these critically endangered species.     

Operator Independent Focused High Frequency ISM Band for Fat Reduction: Porcine Model

Background

Selective fat reduction has been clearly shown for various methods and energy modalities including cryolipolysis and high intensity focused thermal ultrasound. Mathematical modeling of focused high frequency of the EM spectrum has indicated that selective heating of fat is possible using wavelengths not previous explored. The purpose of this study was to demonstrate in the porcine model that selective heating of fat is possible with a non‐contact, operator independent device.

Methods

High frequencies of the Industrial, Scientific and Medical (ISM) RF band were utilized to reduce abdominal fat in a porcine model. Practical application of mathematical modeling allowed an auto‐feedback loop to be developed to allow operator independent adjustment of energy to maintain subcutaneous fat at 45–46°C while overlying skin remained at 40–41°C.

Results

Treatments of three Vietnamese pigs were performed under anesthesia in a certified veterinary facility. Gross and microscopic histologic results demonstrated a marked reduction in adipocytes of the treated area after 4 treatments of a total of 30 minutes each, with incremental fat diminution after each treatment. A final 70% reduction of the abdominal fat layer was seen in the treated areas. Duplex ultrasound revealed a reduction of fat layer from 7.6 to 2.9 mm. Histologic evaluation revealed that epidermis, dermis, and adnexal structures such as hair follicles were unaffected by the treatment, while adipocytes were significantly affected.

Conclusion

A new model of fat reduction using high frequency RF has been successfully achieved in a porcine model. This has very positive implications in the development of an operator independent, contact free device for reduction of fat in clinical practice. Lasers Surg. Med. © 2013 Wiley Periodicals, Inc.     

Plasma Cytokine Analysis in Patients with Advanced Extremity Melanoma Undergoing Isolated Limb Infusion

Background

Preprocedure clinical and pathologic factors have failed to consistently differentiate complete response (CR) from progressive disease (PD) in patients after isolated limb infusion (ILI) with melphalan for unresectable in-transit extremity melanoma.

Methods

Multiplex immunobead assay technology (Milliplex MAP Human Cytokine/Chemokine Magnetic Bead Panel, Millipore Corp., Billerica, MA; and Magpix analytical test instrument, Luminex Corp., Austin, TX) was performed on pre-ILI plasma to determine concentrations of selected cytokines (MIP-1α, IL-1Rα, IP-10, IL-1β, IL-1α, MCP-1, IL-6, IL-17, EGF, IL-12p40, VEGF, GM-CSF, and MIP-1β) on a subset of patients (n = 180) who experienced CR (n = 23) or PD (n = 24) after ILI. Plasma from normal donors (n = 12) was also evaluated.

Results

Of 180 ILIs performed, 28 % (95 % confidence interval 22–35, n = 50) experienced a CR, 14 % (n = 25) experienced a partial response, 11 % (n = 21) had stable disease, 34 % (n = 61) had PD, and 13 % (n = 23) were not evaluable for response. Tumor characteristics and pharmacokinetics appeared similar between CR (n = 23) and PD (n = 24) patients who underwent cytokine analysis. Although there were no differences in cytokine levels between CR and PD patients, there were differences between the melanoma patients and controls. MIP-1α, IL-1Rα, IL-1β, IL-1α, IL-17, EGF, IL-12p40, VEGF, GM-CSF, and MIP-1β were significantly higher in normal controls compared to melanoma patients, while IP-10 was lower (p < 0.001) in controls compared to melanoma patients.

Conclusions

Patients with unresectable in-transit melanoma appear to have markedly decreased levels of immune activating cytokines compared to normal healthy controls. This further supports a potential role for immune-targeted therapies and immune monitoring in patients with regionally advanced melanoma.     

Enhancement of protein expression by alphavirus replicons by designing self-replicating subgenomic RNAs

Since the development of infectious cDNA clones of viral RNA genomes and the means of delivery of the in vitro-synthesized RNA into cells, alphaviruses have become an attractive system for expression of heterologous genetic information. Alphaviruses replicate exclusively in the cytoplasm, and their genetic material cannot recombine with cellular DNA. Alphavirus genome-based, self-replicating RNAs (replicons) are widely used vectors for expression of heterologous proteins. Their current design relies on replacement of structural genes, encoded by subgenomic RNAs (SG RNA), with heterologous sequences of interest. The SG RNA is transcribed from a promoter located in the alphavirus-specific RNA replication intermediate and is not further amplified. In this study, we have applied the accumulated knowledge of the mechanism of alphavirus replication and promoter structures, in particular, to increase the expression level of heterologous proteins from Venezuelan equine encephalitis virus (VEEV)-based replicons. During VEEV infection, replication enzymes are produced in excess to RNA replication intermediates, and a large fraction of them are not involved in RNA synthesis. The newly designed constructs encode SG RNAs, which are not only transcribed from the SG promoter, but are additionally amplified by the previously underused VEEV replication enzymes. These replicons produce SG RNAs and encoded proteins of interest 10- to 50-fold more efficiently than those using a traditional design. A modified replicon encoding West Nile virus (WNV) premembrane and envelope proteins efficiently produced subviral particles and, after a single immunization, elicited high titers of neutralizing antibodies, which protected mice from lethal challenge with WNV.Alphaviruses are a group of enveloped viruses with a positive-strand RNA genome that replicate in most commonly used cell lines to titers exceeding 10¹⁰ infectious units (inf.u)/mL (1, 2). Upon infection, the genomic RNA serves as a template for translation of viral nonstructural proteins that form replication complexes (3). Within a few hours postinfection, these complexes synthesize large amounts of viral genomic and subgenomic (SG) RNA (3). The SG RNA is transcribed from the SG promoter and serves as a template for translation of viral structural proteins: capsid, E2 and E1, which ultimately assemble with genomic RNA into infectious viral particles. This highly efficient virus-specific RNA and protein synthesis, coupled with the availability of infectious cDNA clones, have made alphaviruses an attractive system for designing self-replicating vectors for delivery and expression of heterologous genetic information. The most widely used alphavirus-based expression systems are based on replacement of viral structural genes by a gene(s) of interest (4). These modified viral genomes, termed replicons, can be synthesized in vitro and delivered into cells either by transfection or in infectious viral particles, which deliver essentially every packaged RNA molecule into the cells both in vivo and in vitro.In recent years, significant progress has been made in our understanding of the mechanism of alphavirus replication. Detailed studies have elucidated the structure and function of the RNA promoters, critical aspects of virus–host cell interactions, and the composition of the replication complexes (5–12). These mechanistic studies of alphavirus replication raised the question of whether we are using their entire expression potential, and whether the traditional replicon design can be further improved to achieve higher levels of heterologous protein production. In this project, we sought to apply the latest advances in understanding of alphavirus RNA replication to design a new generation of Venezuelan equine encephalitis virus (VEEV) genome-based expression systems. The distinguishing feature of these constructs is the modification of the SG RNAs. These SG RNAs have been engineered to contain the cis-acting promoter elements, which are normally present at the 5′ end of the viral genome and mediate genomic RNA replication (8, 13, 14). Thus, in these newly designed VEEV replicons (VEErep), the SG RNAs were not only transcribed from the SG promoter, but were capable of replication/amplification by the VEEV replication complexes. As a result, the heterologous gene expression was more efficient than that of the existing constructs, which use replicons with the standard SG RNAs. The expression level of heterologous protein encoded by the improved replicons was also found to be dependent on coexpression of VEEV capsid protein. The VEEV replicons, which use both amplification of the SG RNA and express capsid protein, provide a platform for development of a variety of more efficient expression systems and have numerous applications. To illustrate this, we have generated a VEEV replicon encoding the premembrane and envelope (prM/E) proteins of West Nile virus (WNV). Particles containing the newly designed replicons induced high levels of WNV E protein expression in vitro and elicited robust protective immunity in mice.     

HBeAg and anti-HBe detection by radioimmunoassay: correlation with vertical transmission of hepatitis B virus in Taiwan.

A sensitive radioimmunoassay technique was used to detect hepatitis B e antigen (HBeAg). A strong correlation was found between HBeAg positivity of the serum of hepatitis B surface antigen (HBsAg) carrier women in Taiwan and the subsequent development of surface antigenemia in their babies. All babies who became chronic HBsAg carriers were born to HBeAg positive women, maternal HBeAg positivity being a better prior indication of chronic antigenemia developing in the baby than the HBsAg titer in the mother's serum.     

The effect of respiratory care department management of a blood gas analyzer on the appropriateness of arterial blood gas utilization

We investigated the effects of establishing a blood gas analysis service controlled by respiratory care practitioners (RCPs) on the appropriateness of arterial blood gas (ABG) sampling. An ABG analyzer was placed outside the surgical intensive care unit (SICU) and only RCPs were permitted to process samples on it. In 1-month and 1-year follow-up audits of appropriateness of ABG analysis, the nursing staff improved from 42% appropriate to 73% appropriate in both follow-up periods. RCPs maintained a high degree of appropriateness in all periods (90%, 87%, and 91%), although the percentage of the total ABGs performed by RCPs increased. Additional benefits included a better mutual understanding of each caregiver's role and work load, more collaboration among caregiver groups, and caregiver's perception of improved patient care.