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Peptide nanoassemblies have garnered remarkable importance in the development of novel nanoscale biomaterials for drug delivery into tumor cells. Taking advantage of receptor mediated recognition of two known peptides, angiopep-2 (TFFYGGSRGKRNNFKTEEY) and A-COOP-K (ACGLSGLC10 VAK) that bind to the over-expressed receptors low density lipoprotein (LRP-1) and fatty acid binding protein (FABP3) respectively, we have developed new peptide conjugates by combining the anti-inflammatory, antitumor compound azelaic acid with angiopep-2, which efficiently self-assembled into nanofibers. Those nanofibers were then functionalized with the A-COOP-K sequence and formed supramolecular hierarchical structures that were found to entrap the chemotherapeutic drug doxorubicin efficaciously. Furthermore, the nanoassemblies were found to release the drug in a dose-dependent manner and showed a stepwise increase over a period of 2 weeks under acidic conditions. Two cell lines (U-87-MG and U-138-MG) were utilized as models for glioblastoma cells grown in the presence of serum and under serum-free conditions to mimic the growth conditions of natural tumors. The drug entrapped assemblies were found to inhibit the cell proliferation of both U-87 and U-138MG glioblastoma cells. Three dimensional spheroids of different sizes were grown to mimic the tumors and evaluate the efficacy of drug release and internalization. Our results indicated that the nanoassemblies were found to have higher internalization of DOX and were well-spread throughout the spheroids grown, particularly under serum-free conditions. The nanoassemblies also displayed blood-brain barrier penetration when tested with a multicellular in vitro model. Such self-assembled nanostructures with targeting ability may provide a suitable platform for the development of new peptide-based biomaterials that can provide more insights about the mechanistic approach for drug delivery for not only 2D cell cultures but also 3D tumoroids that mimic the tumor microenvironments., Competing Interests: The authors declare no competing financial interest., (© 2023 The Authors. Published by American Chemical Society.)

Introduction: Urinalysis is the defining test for microscopic hematuria and has traditionally been performed via microscopy. In recent years automated urinalysis machines, which use different technology to identify cells, have been widely adopted. There is little understanding of these machines in the urology community and how their use may affect evaluation of hematuria., Methods: We compared commercially available automated urinalysis machines and reviewed the literature comparing these machines to microscopic urinalysis. We also reviewed local hospital practices regarding these systems., Results: We identified 6 commercially available machines, almost all of which use flow cytometry as the mechanism to identify red blood cells. Review of the literature and local practice revealed differing definitions of normal range for red blood cells, most of which do not correlate with the American Urological Association definition of microscopic hematuria. There are also several methods for calibrating the machines in comparison to traditional microscopy., Conclusions: Use of automated urinalysis machines is widespread. However, these methods do not correspond exactly to traditional microscopy and use varying definitions of microscopic hematuria. These findings warrant additional investigation into the role of these devices in the definition of microscopic hematuria to prevent unneeded evaluations and to appropriately use health care resources.

Super-selective vascular control prior to robotic partial nephrectomy (also known as 'zero-ischemia') is a novel surgical technique that promises to reduce warm ischemia time. The technique has been shown to be feasible but adds substantial technical complexity and cost to the procedure. We present a simplified retrograde dissection of the renal hilum to achieve selective vascular control during robotic partial nephrectomy. Consecutive patients with stage 1 solid and complex cystic renal masses underwent robotic partial nephrectomies with selective vascular control using a modification to previously described super-selective robotic partial nephrectomy. In each case, the renal arterial branch supplying the mass and surrounding parenchyma was dissected in a retrograde fashion from the tumor. Intra-renal dissection of the interlobular artery was not performed. Intra-operative immunofluorescence was not utilized as assessment of parenchymal ischemia was documented before partial nephrectomy. Data was prospectively collected in an IRB-approved partial nephrectomy database. Operative variables between patients undergoing super-selective versus standard robotic partial nephrectomy were compared. Super-selective partial nephrectomy with retrograde hilar dissection was successfully completed in five consecutive patients. There were no complications or conversions to traditional partial nephrectomy. All were diagnosed with renal cell carcinoma and surgical margins were all negative. Estimated blood loss, warm ischemia time, operative time and length of stay were all comparable between patients undergoing super-selective and standard robotic partial nephrectomy. Retrograde hilar dissection appears to be a feasible and safe approach to super-selective partial nephrectomy without adding complex renovascular surgical techniques or cost to the procedure.

Disodium-fosfomycin pharmacokinetics has been studied in different species after oral, intravenous, intramuscular and subcutaneous administration. At present there are neither documented clinical experiences of the use of fosfomycin in pigs nor any published studies in weaning piglets, although it is a period of high incidence of infectious diseases. The pharmacokinetics and the bioavailability of sodium fosfomycin were studied in post weaning piglets after intravenous and intramuscular administration of 15 mg/kg of body weight. Plasma concentrations were measured by a high-performance liquid ms/ms. After IV administration the area under the fosfomycin concentration:time curve in plasma was AUC(0-12) of 120.00 ± 23.12 μg h/ml and the volume of distribution (Vd) of 273.00 ± 40.70 ml/kg. The elimination was rapid with a plasma clearance of 131.50 ± 30.07 ml/kg/h and a T(1/2) of 1.54 ± 0.40 h. Peak serum concentration (Cmax), Tmax, AUC(0-12) and bioavailability for the IM administration were 43.00 ± 4.10 μg/ml, 0.75 ± 0.00 h, 99.00 ± 0.70 μg h/ml and 85.5 ± 9.90% respectively. Different authors have determined a minimum inhibitory concentration (MIC90) ranging from 0.25 μg/ml for Streptococcus sp. and 0.5 μg/ml for Escherichia coli. Considering the above, and according to the values of plasma concentration vs time profiles observed in this study, effective plasma concentrations of fosfomycin for sensitive bacteria can be obtained following IV and IM administration of 15 mg/kg in piglets., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Purpose: To validate the feasibility of robot-assisted simple retropubic prostatectomy (RSP) for men with severe benign prostatic hyperplasia (>80 g)., Patients and Methods: Institutional Review Board approval was not sought for this series. Men were offered RSP by two surgeons with a combined experience of >350 robot-assisted radical prostatectomies. The RSP replicated previously published robotic and laparoscopic techniques. Postoperative management consisted of continuous bladder irrigation and closed suction pelvic drainage without suprapubic catheterization., Results: A total of nine men were treated. Indications for RSP included urinary retention in three patients, failed medical management in eight patients, and refusal of medical management in one. Average age was 68 years, mean prostate-specific antigen level was 17.4 ng/mL, and the average preoperative gland size (height-width-length volume) was 136.5 g (range 86-265 g). No operative or immediate postoperative complications occurred, and no transfusions were needed. Average blood loss, operative time, and console time were 206 mL, 183 minutes, and 147 minutes, respectively. Average pathologic adenoma volume was 112 g (range 53-220 g). Average hospitalization time and catheterization time were 32 hours and 13 days, respectively. The mean preoperative International Prostate Symptom Score was 17.8 compared with 7.77 at 6 months postoperatively (P=0.0096, 95% CI 2.83 - 17.40), with a mean follow-up time of 9.25 months. The mean Sexual Health Inventory for Men score was 12.7 preoperatively compared with 12.5 postoperatively (P=0.74, 95% confidence interval - 6.66-9.16). Persistent, severe urinary incontinence (4-6 pads per day) occurred in one patient., Conclusions: RSP is safe and reproducible when performed by experienced robotic surgeons and provides similar benefits to those associated with robot-assisted radical prostatectomy. In our limited experience, hemostasis was markedly decreased when compared with the open technique. Further investigation is necessary before widespread application of RSP.

It has recently become clear that the transcriptional output of the human genome is far more abundant than previously anticipated, with the vast majority of transcripts not coding for protein. Utilizing whole-genome tiling arrays, we analyzed the transcription across the entire genome in both normal human bronchial epithelial cells (NHBE) and NHBE cells exposed to the tobacco carcinogen NNK. Our efforts focused on the characterization of non-coding transcripts that were greater than 300 nucleotides in length and whose expression was increased in response to NNK. We identified 12 Long Stress-Induced Non-coding Transcripts that we term LSINCTs. Northern blot analysis revealed that these transcripts were larger than predicted from the tiling array data. Quantitative real-time RT-PCR performed across a panel of normal cell lines indicates that these transcripts are more abundantly expressed in rapidly growing tissues or in tissues that are more prone to cellular stress. These transcripts that have increased expression after exposure to NNK also had increased expression in a number of lung cancer cell lines and also in many breast cancer cell lines. Collectively, our results identified a new class of long stress responsive non-coding transcripts, LSINCTs, which have increased expression in response to DNA damage induced by NNK. LSINCTs interestingly also have increased expression in a number of cancer-derived cell lines, indicating that the expression is increased in both, correlating cellular stress and cancer., (Copyright 2010 Elsevier Inc. All rights reserved.)

We describe the genome sequencing of an anonymous individual of African origin using a novel ligation-based sequencing assay that enables a unique form of error correction that improves the raw accuracy of the aligned reads to >99.9%, allowing us to accurately call SNPs with as few as two reads per allele. We collected several billion mate-paired reads yielding approximately 18x haploid coverage of aligned sequence and close to 300x clone coverage. Over 98% of the reference genome is covered with at least one uniquely placed read, and 99.65% is spanned by at least one uniquely placed mate-paired clone. We identify over 3.8 million SNPs, 19% of which are novel. Mate-paired data are used to physically resolve haplotype phases of nearly two-thirds of the genotypes obtained and produce phased segments of up to 215 kb. We detect 226,529 intra-read indels, 5590 indels between mate-paired reads, 91 inversions, and four gene fusions. We use a novel approach for detecting indels between mate-paired reads that are smaller than the standard deviation of the insert size of the library and discover deletions in common with those detected with our intra-read approach. Dozens of mutations previously described in OMIM and hundreds of nonsynonymous single-nucleotide and structural variants in genes previously implicated in disease are identified in this individual. There is more genetic variation in the human genome still to be uncovered, and we provide guidance for future surveys in populations and cancer biopsies.

Both arsenic and benzo[a]pyrene (BaP) inhibit terminal differentiation and alter growth potential in normal human epidermal keratinocytes (NHEK) in vitro. To identify molecular alterations that may be involved in these cellular processes, microarray analysis was carried out on NHEK treated with BaP or arsenic. The gene expression microarray results measuring mRNA levels were as follows: (1) in total, the expression of 85 genes was induced and 17 genes was suppressed by 2.0 microm BaP. (2) Arsenic at an equitoxic dose (5.0 microm) induced the expression of 106 and suppressed 15 genes. Quantitative real-time RT-PCR was used subsequently to confirm microarray findings on selected genes involved in keratinocyte growth and differentiation pathways. These studies confirmed increased mRNA levels in NHEK by BaP of alpha-integrin binding protein 63 (AIBP63) (2.48-fold), retinoic acid- and interferon-inducible protein (IFIT5) (2.74-fold), interleukin-1 alpha (IL1A) (2.64-fold), interleukin-1 beta (IL1B) (2.84-fold) and Ras guanyl releasing protein 1 (RASGRP1) (3.14-fold). Real-time RT-PCR confirmed that arsenic increased mRNA levels of the following genes: retinoblastoma 1 (RB1) (5.4-fold), retinoblastoma-binding protein 1 (ARID4A) (6.8-fold), transforming growth factor beta-stimulated protein (TSC22D1) (6.84-fold), MAX binding protein (MNT) (2.44-fold), and RAD50 (4.24-fold). Collectively, these results indicate that these chemicals target different genes and molecular pathways involved in the regulatory processes controlling NHEK proliferation and differentiation. Mechanistic studies with a subset of genes may allow the correlation of alterations in these molecular markers with chemical-specific blocks to differentiation in NHEK., (Copyright (c) 2007 John Wiley & Sons, Ltd.)

Recent studies with tiling arrays have revealed more genomic transcription than previously anticipated. Whole new groups of non-coding transcripts (NCTs) have been detected. Some of these NCTs, including miRNAs, can regulate gene expression. To date, most known NCTs studied have been relatively short, but several important regulatory NCTs, including XIST, MALAT-1, BC1 and BC200, are considerably larger in length and represent a novel class of long, non-coding RNA species. Whole-genome tiling arrays were utilized to identify novel long NCTs across the entire human genome. Our results have identified a new group of long (>400 nt), abundantly expressed NCTs and have found that a subset of these are also highly evolutionarily conserved. In this report, we have begun to characterize 15 long, conserved NCTs. Quantitative real-time RT-PCR was used to analyze their expression in different normal human tissue and also in breast and ovarian cancers. We found altered expression of many of these NCTs in both cancer types. In addition, several of these NCTs have consistent mutations when sequences of normal samples were compared with a panel of cancer-derived cell lines. One NCT was found to be consistently mutated in a panel of endometrial cancers compared with matched normal blood. These NCTs were among the most abundantly expressed transcripts detected. There are probably many long, conserved NCTs, albeit with lower levels of expression. Although the function of these NCTs is currently unknown, our study indicates that they may play an important function in both normal cells and in cancer development.

Common fragile sites (CFS) are large, genomically unstable regions, which are hot-spots for deletions and other alterations, especially in cancer cells. Several have been shown to contain genes that span large genomic regions, such as FHIT (1.5 Mb), WWOX (1.0 Mb), GRID2 (1.36 Mb), PARK2 (1.3 Mb), and RORA (730 kb). These genes are frequently inactivated in multiple different cancers, and FHIT and WWOX are shown to function as tumor suppressors. The disabled-1 gene (DAB1) is one of the human homologs of the Drosophila disabled locus, which in mammals is involved in neuronal migration and lamination in the developing cerebral cortex. Mice DAB1 inactivation results in the neurological mutant Scrambler, having similarities to mice with the inactivation of PARK2 (Quaker), GRID2 (Lurcher), and RORA (Staggerer). We were interested in whether DAB1 was another large CFS gene that could have cancer development importance. We demonstrated here that the human DAB1 gene (spanning 1.25 Mb) mapped within FRA1B CFS region on chromosomal band 1p32.2. Real-time RT-PCR analysis revealed that the expression level of DAB1 was decreased in many human cancer samples, including primary tumor tissues and cancer-derived cell lines, from several different cancers, especially in brain and endometrial cancer. Additionally, the introduction of an over-expression DAB1 plasmid into two different cell lines, having insignificant endogenous DAB1 expression, resulted in decreased cell growth. In summary, DAB1 is another gene that resides within an unstable CFS region and might play a role in human tumorigenesis. These data may provide further linkage between neurological development and cancer., ((c) 2007 Wiley-Liss, Inc.)

The common fragile sites (CFSs) are large regions of profound genomic instability found in all individuals. A number of the CFSs have been found to span genes that extend over large genomic regions (>700 kb). The expression of these genes is frequently abrogated in a number of different cancers and several of them have already been shown to function as tumor suppressor genes, both in vitro and in vivo. We analyzed the expression of 14 large CFS genes in two distinct groups of head and neck cancers using real-time RT-PCR. The first were oral tongue squamous cell carcinomas (SCCs) and the second were base of tongue/tonsillar (oropharyngeal) SCCs. These two groups were previously examined for the presence of human papillomavirus (HPV) and while 46% of the oropharyngeal cancers were positive for HPV16 only one of 52 oral cancers contained HPV16 sequences. We observed a distinct pattern of loss of expression of the large CFS genes in the two groups of head and neck cancers. In addition, there was no correlation between the relative instability in different CFS regions and which genes were inactivated. Thus, this report demonstrates another distinction between these two groups of head and neck cancer. In addition, it suggests that there is selection for loss of expression of specific CFS genes in these cancers., (Copyright 2008 S. Karger AG, Basel.)

The common fragile sites are large regions of genomic instability that are found in all individuals and are hot spots for chromosomal rearrangements and deletions. A number of the common fragile sites have been found to span genes that are encoded by very large genomic regions. Two of these genes, FHIT and WWOX, have already been demonstrated to function as tumor suppressors. In this review we will discuss the large common fragile site genes that have been identified to date, and the role that these genes appear to play both in cellular responses to stress and in the development of cancer.

Common fragile sites (CFSs) are large regions of profound genomic instability found in all individuals. Spanning the center of the two most frequently expressed CFS regions, FRA3B (3p14.3) and FRA16D (16q23.2), are the 1.5 Mb FHIT gene and the 1.0 Mb WWOX gene. These genes are frequently deleted and/or altered in many different cancers. Both FHIT and WWOX have been demonstrated to function as tumor suppressors, both in vitro and in vivo. A number of other large CFS genes have been identified and are also frequently inactivated in multiple cancers. Based on these data, several additional very large genes were tested to determine if they were derived from within CFS regions, but DCC and RAD51L1 were not. However, the 2.0 Mb DMD gene and its immediately distal neighbor, the 1.8 Mb IL1RAPL1 gene are CFS genes contained within the FRAXC CFS region (Xp21.2-->p21.1). They are abundantly expressed in normal brain but were dramatically underexpressed in every brain tumor cell line and xenograft (derived from an intracranial model of glioblastoma multiforme) examined. We studied the expression of eleven other large CFS genes in the same panel of brain tumor cell lines and xenografts and found reduced expression of multiple large CFS genes in these samples. In this report we show that there is selective loss of specific large CFS genes in different cancers that does not appear to be mediated by the relative instability within different CFS regions. Further, the inactivation of multiple large CFS genes in xenografts and brain tumor cell lines may help to explain why this type of cancer is highly aggressive and associated with a poor clinical outcome., (Copyright (c) 2008 S. Karger AG, Basel.)

The common fragile sites are regions of profound genomic instability found in all individuals. The full size of each region of instability ranges from under one megabase (Mb) to greater than 10 Mbs. At least half of the CFS regions have been found to span extremely large genes that spanned from 600 kb to greater than 2.0 Mbs. The large CFS genes are also very interesting from a cancer perspective as several of them, including FHIT and WWOX, have already demonstrated the capacity to function as tumor suppressor genes, both in vitro and in vivo. We estimate that there may be 40-50 large genes localized in CFS regions. The expression of a number of the large CFS genes has been previously shown to be lost in many different cancers and this is frequently associated with a worse clinical outcome for patients. To determine if there was selection for the inactivation of different large CFS genes in different cancers, we examined the expression of 13 of the 20 known large CFS genes: FHIT, WWOX, PARK2, GRID2, NBEA, DLG2, RORA isoforms 1 and 4, DAB1, CNTNAP2, DMD, IL1RAPL1, IMMP2L and LARGE in breast, ovarian, endometrial and brain cancers using real-time RT-PCR analysis. Each cancer had a distinct profile of different large CFS genes that were inactivated. Interestingly, in breast, ovarian and endometrial cancers there were some cancers that had inactivation of expression of none or only one of the tested genes, while in other specimens there was inactivation of multiple tested genes. Brain cancers had inactivation of many of the tested genes, a number of which function in normal neurological development. We find that there is no relationship between the frequency that any specific CFS is expressed and the frequency that the gene from that region is inactivated in different cancers. Instead, it appears that different cancers select for the inactivation of different large CFS genes., (Copyright (c) 2007 S. Karger AG, Basel.)

Normal human epidermal keratinocytes (NHEK) have been chosen as an in vitro model to test the hypothesis that chemicals which alter or interfere in cellular differentiation will concomitantly induce growth perturbations and are, thus, potential carcinogens. In these studies, we have focused on two known skin carcinogens, arsenic and benzo(a)pyrene (BaP). Our results demonstrated that BaP inhibits terminal differentiation in NHEK, as measured by cross-linked envelope (CLE) formation, up to 5.8-fold in control and 1.7-fold in calcium (Ca2+)-treated cells. In comparison, arsenic decreased CLE formation 20-fold in control cells and 5.5-fold in Ca2+-treated NHEK. To characterize the effects of these agents on the growth rate and cell cycle distributions of NHEK, flow cytometric analysis was used. BaP at 2 microM increased proliferation rates by 29%. Altered cell-cycle distribution in BaP-treated cells indicated a more rapid progression through the cell cycle, possibly by a shortened G2 phase. In contrast, arsenic at 5 microM inhibited proliferation by 25%; growth arrest (9%) was also observed in NHEK treated with 2 mM Ca2+. Our findings suggest that, although both BaP and arsenic inhibit CLE production in NHEK, different mechanisms may be involved. Studies in progress will attempt to identify molecular markers involved in the observed chemical effects. These markers will facilitate a mechanistic understanding of how an altered balance between growth and differentiation may play a role in the transformation process in NHEK.