Construction of Canine Herpesvirus Vector Expressing Foreign Genes Using a lacZ-TK Gene Cassette as a Double Selectional Marker

An improved method for constructing canine herpesvirus (CHV) recombinants expressing foreign genes by using the lacZ-TK gene cassette as a double selectional marker was developed. A recombinant CHV carrying the lacZ-TK gene at a targeted gene locus was constructed and used as a parental virus for generating new recombinants. The parental virus formed blue plaques and was sensitive to TK-specific drugs, while newly generated recombinants, in which the lacZ-TK gene was replaced with the desired foreign gene, become both resistant to the TK-specific drugs and formed white plaques. Recombinants were isolated by using the combination of drug selection and color selection. This improved method allows construction of recombinant CHV with great ease, because the drug selection can enrich the frequency of recombinant CHV from 0.01–0.1% to 10–80%. This method was employed to construct a recombinant CHV that expressed rabies virus (RV) glycoprotein (G protein). This revised version was published online in July 2006 with corrections to the Cover Date.     

Can random bladder biopsies be eliminated after bacillus Calmette–Guérin therapy against carcinoma in situ?

Purpose

Intravesical bacillus Calmette–Guérin (BCG) is the standard of care for bladder carcinoma in situ (CIS). The response to BCG therapy against CIS is generally assessed by random bladder biopsy (RBB). In this study, we examined the necessity of routine RBB after BCG therapy.

Methods

We retrospectively identified 102 patients who were initially diagnosed with CIS with or without papillary tumor and received subsequent 6–8-week BCG therapy. Thereafter, all patients underwent voiding cytology analysis, cystoscopy, and RBB to evaluate the effects of BCG therapy. We evaluated the association between clinical parameters (voiding cytology and cystoscopy findings) and the final pathological results by RBB specimens.

Results

According to the pathological results of RBB, 30 (29%) patients had BCG-unresponsive disease (remaining urothelial carcinoma was confirmed pathologically) and 20 were diagnosed with CIS. Positive/suspicious voiding cytology and positive cystoscopy findings were well observed in patients who had BCG-unresponsive disease compared with their counterparts (p?=?0.116, and p?<?0.001, respectively). The sensitivity (Sen.), specificity (Spe.), positive predictive value (PPV), and negative predictive value (NPV) of voiding cytology were 50%, 68%, 39%, and 77%, respectively. The values for cystoscopy findings were as follows: Sen.: 87%, Spe.: 57%, PPV: 46%, and NPV: 91%. The values for their combination (having either of them) were as follows: Sen.: 100%, Spe.: 44%, PPV: 43%, and NPV: 100%.

Conclusion

RBB after BCG therapy for patients with negative voiding cytology and negative cystoscopy may be omitted because their risk of BCG-unresponsive disease is significantly low (NPV: 100%).

     

All-in-One Silk Fibroin Sponge as the Vitrification Cryodevice of Rat Pancreatic Islets and the VEGF-Embedded Scaffold for Subrenal Transplantation

Islet transplantation is a promising option for the clinical treatment of insulin-dependent diabetes, but a reliable islet cryopreservation/transplantation protocol should be established to overcome the donor shortage. The current study reports that a silk fibroin (SF) sponge disk can be used as a cryodevice for vitrification of large quantity pancreatic islets and the scaffold for subsequent subrenal transplantation in a rat model. The marginal islet mass (550 islet equivalents [IEQs]) on an SF sponge disk was vitrified-warmed and transplanted beneath the kidney capsule of a streptozotocin-induced diabetic rat with or without vascular endothelial growth factor (VEGF). Subrenal transplantation (no scaffold) of 550 IEQ fresh islets and post-warm islets vitrified on a nylon mesh device resulted in achieving euglycemia of recipient rats at 60% and 0%, respectively. Transplantation of 550 IEQ islets vitrified-warmed on an SF sponge disk failed to achieve euglycemia of recipient rats (0%), but the VEGF inclusion in the SF sponge disk contributed to acquiring the euglycemic recipients (33%). All cured recipient rats regained hyperglycemia after nephrectomy, and the histopathologic analysis exhibited a well-developing blood vessel network into the islet engrafts. Thus, an SF sponge disc was successively available as the cryodevice for islet vitrification, the transporter of the angiogenic VEGF, and the scaffold for subrenal transplantation in the rat model.     

Analysis of acute-phase toxicities of intensity-modulated proton therapy using a model-based approach in pharyngeal cancer patients

Pharyngeal cancer patients treated with intensity-modulated proton therapy (IMPT) using a model-based approach were retrospectively reviewed, and acute toxicities were analyzed. From June 2016 to March 2019, 15 pharyngeal (7 naso-, 5 oro- and 3 hypo-pharyngeal) cancer patients received IMPT with robust optimization. Simulation plans for IMPT and intensity-modulated X-ray therapy (IMXT) were generated before treatment. We also reviewed 127 pharyngeal cancer patients with IMXT in the same treatment period. In the simulation planning comparison, all of the normal-tissue complication probability values for dysphagia, dysgeusia, tube-feeding dependence and xerostomia were lower for IMPT than for IMXT in the 15 patients. After completing IMPT, 13 patients completed the evaluation, and 12 of these patients had a complete response. The proportions of patients who experienced grade 2 or worse acute toxicities in the IMPT and IMXT cohorts were 21.4 and 56.5% for dysphagia (P < 0.05), 46.7 and 76.3% for dysgeusia (P < 0.05), 73.3 and 62.8% for xerostomia (P = 0.43), 73.3 and 90.6% for mucositis (P = 0.08) and 66.7 and 76.4% for dermatitis (P = 0.42), respectively. Multivariate analysis revealed that IMPT was independently associated with a lower rate of grade 2 or worse dysphagia and dysgeusia. After propensity score matching, 12 pairs of IMPT and IMXT patients were selected. Dysphagia was also statistically lower in IMPT than in IMXT (P < 0.05). IMPT using a model-based approach may have clinical benefits for acute dysphagia.     

Nondomain biopolymers: Flexible molecular strategies to acquire biological functions

A long-standing assumption in molecular biology posits that the conservation of protein and nucleic acid sequences emphasizes the functional significance of biomolecules. These conserved sequences fold into distinct secondary and tertiary structures, enable highly specific molecular interactions, and regulate complex yet organized molecular processes within living cells. However, recent evidence suggests that biomolecules can also function through primary sequence regions that lack conservation across species or gene families. These regions typically do not form rigid structures, and their inherent flexibility is critical for their functional roles. This review examines the emerging roles and molecular mechanisms of “nondomain biomolecules,” whose functions are not easily predicted due to the absence of conserved functional domains. We propose the hypothesis that both domain- and nondomain-type molecules work together to enable flexible and efficient molecular processes within the highly crowded intracellular environment.     

A rapid method for detecting barbiturates in serum using EI-SIM

A simple and rapid method for analysis of barbiturates in serum has been developed. In order to extract and clean barbiturates in serum, a separation column packed with Extrelut and Florisil was used, and the eluate was directly analyzed by means of electron impact selected ion monitoring (EI-SIM). Selected ions used were base peak ions of 10 barbituartes, and the internal standard used was allobarbital or secobarbital. The calibration curves were linear over the range 0.5–5 ng. Extraction of replicate serum samples containing 20 g/1.5 ml and 5 g/1.5 ml resulted in a recovery of 87.2–105.2% and 81.6–104.6%, respectively, with the exception of phenobarbital, which was 151.9% and 172.1%, respectively. Secobarbital was also analyzed in the serum of 13 patients who had been given secobarbital intravenously. In 3 out of 10 cases, Secobarbital levels greater than 1 g/ml were detected more than 72 h after administration. This method seems to have possibilities for clinical use.Paper presented at the 2nd International Symposium ADVANCES IN LEGAL MEDICINE, Berlin, Germany, August 30–September 1, 1993