CRISPR-Cas9-mediated barcode insertion into Bacillus thuringiensis for surrogate tracking.

The use of surrogate organisms can enable researchers to safely conduct research on pathogens and in a broader set of conditions. Being able to differentiate between the surrogates used in the experiments and background contamination as well as between different experiments will further improve research efforts. One effective approach is to introduce unique genetic barcodes into the surrogate genome and track their presence using the quantitative polymerase chain reaction (qPCR). In this report, we utilized the CRISPR-Cas9 methodology, which employs a single plasmid and a transformation step to insert five distinct barcodes into Bacillus thuringiensis, a well-established surrogate for Bacillus anthracis when Risk Group 1 organisms are needed. We subsequently developed qPCR assays for barcode detection and successfully demonstrated the stability of the barcodes within the genome through five cycles of sporulation and germination. Additionally, we conducted whole-genome sequencing on these modified strains and analyzed 187 potential Cas9 off-target sites. We found no correlation between the mutations observed in the engineered strains and the predicted off-target sites, suggesting this genome engineering strategy did not directly result in off-target mutations in the genome. This simple approach has the potential to streamline the creation of barcoded B. thuringiensis strains for use in future studies on surrogate genomes. IMPORTANCE: The use of Bacillus anthracis as a biothreat agent poses significant challenges for public health and national security. Bacillus anthracis surrogates, like Bacillus thuringiensis, are invaluable tools for safely understanding Bacillus anthracis properties without the safety concerns that would arise from using a virulent strain of Bacillus anthracis. We report a simple method for barcode insertion into Bacillus thuringiensis using the CRISPR-Cas9 methodology and subsequent tracking by quantitative polymerase chain reaction (qPCR). Moreover, whole-genome sequencing data and CRISPR-Cas9 off-target analyses in Bacillus thuringiensis suggest that this gene-editing method did not directly cause unwanted mutations in the genome. This study should assist in the facile development of barcoded Bacillus thuringiensis surrogate strains, among other biotechnological applications in Bacillus species.

Exposome and Metabolome Analysis of Sugarcane Workers Reveals Predictors of Kidney Injury.

Introduction: Sugarcane workers are exposed to potentially hazardous agrochemicals, including pesticides, heavy metals, and silica. Such occupational exposures present health risks and have been implicated in a high rate of kidney disease seen in these workers. Methods: To investigate potential biomarkers and mechanisms that could explain chronic kidney disease (CKD) among this worker population, paired urine samples were collected from sugarcane cutters at the beginning and end of a harvest season in Guatemala. Workers were then separated into 2 groups, namely those with or without kidney function decline (KFD) across the harvest season. Urine samples from these 2 groups underwent elemental analysis and untargeted metabolomics. Results: Urine profiles demonstrated increases in silicon, certain pesticides, and phosphorus levels in all workers, whereas heavy metals remained low. The KFD group had a reduction in estimated glomerular filtration rate (eGFR) across the harvest season; however, kidney injury marker 1 did not significantly change. Cross-harvest metabolomic analysis found trends of fatty acid accumulation, perturbed amino acid metabolism, presence of pesticides, and other known signs of impaired kidney function. Conclusion: Silica and certain pesticides were significantly elevated in the urine of sugarcane workers with or without KFD. Future work should determine whether long-term occupational exposure to silica and pesticides across multiple seasons contributes to CKD in these workers. Overall, these results confirmed that multiple exposures are occurring in sugarcane workers and may provide insight into early warning signs of kidney injury and may help explain the increased incidence of CKD among agricultural workers.

Intranasal Administration of Sugarcane Ash Causes Chronic Kidney Disease in Rats.

Background. Silica nanoparticles found in sugarcane ash have been postulated to be a toxicant contributing to chronic kidney disease of unknown etiology (CKDu). However, while the administration of manufactured silica nanoparticles is known to cause chronic tubulointerstitial disease in rats, the effect of administering sugarcane ash on kidney pathology remains unknown. Here we investigate whether sugarcane ash can induce CKD in rats. Methods. Sugarcane ash was administered for 13 weeks into the nares of rats (5 mg/day for 5d/week), and blood, urine and kidney tissues were collected at 13 weeks (at the end of ash administration) and in a separate group of rats at 24 weeks (11 weeks after stopping ash administration). Kidney histology was evaluated, and inflammation and fibrosis (collagen deposition) measured. Results. Sugarcane ash exposure led to the accumulation of silica in the kidneys, lungs, liver and spleen of rats. Mild proteinuria developed although renal function was largely maintained. However, biopsies showed focal glomeruli with segmental glomerulosclerosis, and tubulointerstitial inflammation and fibrosis that tended to worsen even after the ash administration had been stopped. Staining for the lysosomal marker, LAMP-1, showed decreased staining in ash administered rats consistent with lysosomal activation. Conclusion. Sugarcane ash containing silica nanoparticles can cause CKD in rats.

Soft robot-mediated autonomous adaptation to fibrotic capsule formation for improved drug delivery.

The foreign body response impedes the function and longevity of implantable drug delivery devices. As a dense fibrotic capsule forms, integration of the device with the host tissue becomes compromised, ultimately resulting in device seclusion and treatment failure. We present FibroSensing Dynamic Soft Reservoir (FSDSR), an implantable drug delivery device capable of monitoring fibrotic capsule formation and overcoming its effects via soft robotic actuations. Occlusion of the FSDSR porous membrane was monitored over 7 days in a rodent model using electrochemical impedance spectroscopy. The electrical resistance of the fibrotic capsule correlated to its increase in thickness and volume. Our FibroSensing membrane showed great sensitivity in detecting changes at the abiotic/biotic interface, such as collagen deposition and myofibroblast proliferation. The potential of the FSDSR to overcome fibrotic capsule formation and maintain constant drug dosing over time was demonstrated in silico and in vitro. Controlled closed loop release of methylene blue into agarose gels (with a comparable fold change in permeability relating to 7 and 28 days in vivo) was achieved by adjusting the magnitude and frequency of pneumatic actuations after impedance measurements by the FibroSensing membrane. By sensing fibrotic capsule formation in vivo, the FSDSR will be capable of probing and adapting to the foreign body response through dynamic actuation changes. Informed by real-time sensor signals, this device offers the potential for long-term efficacy and sustained drug dosing, even in the setting of fibrotic capsule formation.

Sugarcane ash and sugarcane ash-derived silica nanoparticles alter cellular metabolism in human proximal tubular kidney cells.

Multiple epidemics of chronic kidney disease of an unknown etiology (CKDu) have emerged in agricultural communities around the world. Many factors have been posited as potential contributors, but a primary cause has yet to be identified and the disease is considered likely multifactorial. Sugarcane workers are largely impacted by disease leading to the hypothesis that exposure to sugarcane ash produced during the burning and harvest of sugarcane could contribute to CKDu. Estimated exposure levels of particles under 10 µm (PM10) have been found to be exceptionally high during this process, exceeding 100 µg/m3 during sugarcane cutting and averaging ∼1800 µg/m3 during pre-harvest burns. Sugarcane stalks consist of ∼80% amorphous silica and generate nano-sized silica particles (∼200 nm) following burning. A human proximal convoluted tubule (PCT) cell line was subjected to treatments ranging in concentration from 0.025 µg/mL to 25 µg/mL of sugarcane ash, desilicated sugarcane ash, sugarcane ash-derived silica nanoparticles (SAD SiNPs) or manufactured pristine 200 nm silica nanoparticles. The combination of heat stress and sugarcane ash exposure on PCT cell responses was also assessed. Following 6-48 h of exposure, mitochondrial activity and viability were found to be significantly reduced when exposed to SAD SiNPs at concentrations 2.5 µg/mL or higher. Oxygen consumption rate (OCR) and pH changes suggested significant alteration to cellular metabolism across treatments as early as 6 h following exposure. SAD SiNPs were found to inhibit mitochondrial function, reduce ATP generation, increase reliance on glycolysis, and reduce glycolytic reserve. Metabolomic analysis revealed several cellular energetics pathways (e.g., fatty acid metabolism, glycolysis, and TCA cycle) are significantly altered across ash-based treatments. Heat stress did not influence these responses. Such changes indicate that exposure to sugarcane ash and its derivatives can promote mitochondrial dysfunction and disrupt metabolic activity of human PCT cells.

Safety and Biodistribution of Nanoligomers Targeting the SARS-CoV-2 Genome for the Treatment of COVID-19.

As the world braces to enter its fourth year of the coronavirus disease 2019 (COVID-19) pandemic, the need for accessible and effective antiviral therapeutics continues to be felt globally. The recent surge of Omicron variant cases has demonstrated that vaccination and prevention alone cannot quell the spread of highly transmissible variants. A safe and nontoxic therapeutic with an adaptable design to respond to the emergence of new variants is critical for transitioning to the treatment of COVID-19 as an endemic disease. Here, we present a novel compound, called SBCoV202, that specifically and tightly binds the translation initiation site of RNA-dependent RNA polymerase within the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome, inhibiting viral replication. SBCoV202 is a Nanoligomer, a molecule that includes peptide nucleic acid sequences capable of binding viral RNA with single-base-pair specificity to accurately target the viral genome. The compound has been shown to be safe and nontoxic in mice, with favorable biodistribution, and has shown efficacy against SARS-CoV-2 in vitro. Safety and biodistribution were assessed using three separate administration methods, namely, intranasal, intravenous, and intraperitoneal. Safety studies showed the Nanoligomer caused no outward distress, immunogenicity, or organ tissue damage, measured through observation of behavior and body weight, serum levels of cytokines, and histopathology of fixed tissue, respectively. SBCoV202 was evenly biodistributed throughout the body, with most tissues measuring Nanoligomer concentrations well above the compound KD of 3.37 nM. In addition to favorable availability to organs such as the lungs, lymph nodes, liver, and spleen, the compound circulated through the blood and was rapidly cleared through the renal and urinary systems. The favorable biodistribution and lack of immunogenicity and toxicity set Nanoligomers apart from other antisense therapies, while the adaptability of the nucleic acid sequence of Nanoligomers provides a defense against future emergence of drug resistance, making these molecules an attractive potential treatment for COVID-19.

A Single-Step Digestion for the Quantification and Characterization of Trace Particulate Silica Content in Biological Matrices Using Single Particle Inductively Coupled Plasma-Mass Spectrometry.

The increased use of amorphous silica nanoparticles (SiNPs) in food products, materials science, cosmetics, and pharmaceuticals has raised questions about potential hazards in the environment and in human health. Although SiNPs are generally thought to be benign, recent studies have demonstrated toxicity in different cell and animal models. Despite their ubiquitous use, SiNPs are rarely analyzed quantitatively. Often, the methods used to analyze silicon and SiNPs are difficult, costly, require the use of dangerous reagents, and are prone to interferences. Additionally, characterization of SiNPs in complex matrices requires extensive sample preparation. To address this, we propose a single-step digestion method for the determination of trace SiNP content in biological matrices. For conventional inductively coupled plasma-mass spectrometry (ICP-MS) analysis, biological samples are often digested with concentrated HNO3. We found that with conventional ICP-MS, lower limits of detection (LLOD) of silicon are too high for trace analysis. However, we found that SiNPs are stable at a strong acidic pH; thus, concentrated HNO3 could be used to digest biological samples leaving SiNPs intact. Then, by analysis with single particle ICP-MS, we found that the smallest SiNP that could be read was 185 nm in size. The concentration for the LLOD was found to be 0.032 ppb with interday variability in sizing and concentration at 2.5% and 6.8% respectively. Utilizing this method, SiNPs were accurately sized and counted in cell pellets and media. Our proposed method can be used to accurately quantify and characterize SiNPs (or agglomerated SiNPs) larger than the derived LLOD in a variety of biological matrices and will assist in determining relationships between exposures of SiNPs and toxicity in humans and the environment.

Dynamic actuation enhances transport and extends therapeutic lifespan in an implantable drug delivery platform.

Fibrous capsule (FC) formation, secondary to the foreign body response (FBR), impedes molecular transport and is detrimental to the long-term efficacy of implantable drug delivery devices, especially when tunable, temporal control is necessary. We report the development of an implantable mechanotherapeutic drug delivery platform to mitigate and overcome this host immune response using two distinct, yet synergistic soft robotic strategies. Firstly, daily intermittent actuation (cycling at 1 Hz for 5 minutes every 12 hours) preserves long-term, rapid delivery of a model drug (insulin) over 8 weeks of implantation, by mediating local immunomodulation of the cellular FBR and inducing multiphasic temporal FC changes. Secondly, actuation-mediated rapid release of therapy can enhance mass transport and therapeutic effect with tunable, temporal control. In a step towards clinical translation, we utilise a minimally invasive percutaneous approach to implant a scaled-up device in a human cadaveric model. Our soft actuatable platform has potential clinical utility for a variety of indications where transport is affected by fibrosis, such as the management of type 1 diabetes.

Nanoligomers Targeting Human miRNA for the Treatment of Severe COVID-19 Are Safe and Nontoxic in Mice.

The devastating effects of the coronavirus disease 2019 (COVID-19) pandemic have made clear a global necessity for antiviral strategies. Most fatalities associated with infection from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) result at least partially from uncontrolled host immune response. Here, we use an antisense compound targeting a previously identified microRNA (miRNA) linked to severe cases of COVID-19. The compound binds specifically to the miRNA in question, miR-2392, which is produced by human cells in several disease states. The safety and biodistribution of this compound were tested in a mouse model via intranasal, intraperitoneal, and intravenous administration. The compound did not cause any toxic responses in mice based on measured parameters, including body weight, serum biomarkers for inflammation, and organ histopathology. No immunogenicity from the compound was observed with any administration route. Intranasal administration resulted in excellent and rapid biodistribution to the lungs, the main site of infection for SARS-CoV-2. Pharmacokinetic and biodistribution studies reveal delivery to different organs, including lungs, liver, kidneys, and spleen. The compound was largely cleared through the kidneys and excreted via the urine, with no accumulation observed in first-pass organs. The compound is concluded to be a safe potential antiviral treatment for COVID-19.

Inhaled silica nanoparticles cause chronic kidney disease in rats.

Silica nanoparticles (SiNPs) released during the burning of sugarcane have been postulated to have a role in chronic kidney disease of unknown etiology. We tested the hypothesis that pristine SiNPs of the size present in sugarcane might cause chronic kidney injury when administered through the lung in rats. We administered 200- or 300-nm amorphous SiNPs twice weekly (4 mg/dose), or vehicle by oropharyngeal aspiration for 13 wk to rats followed by euthanasia after an additional 13 wk (26 wk total). Tissues were evaluated for the presence of SiNPs and evidence of histological injury. Both sizes of SiNPs caused kidney damage, with early tubular injury and inflammation (at week 13) that continued to inflammation and chronic fibrosis at week 26 despite discontinuation of the SiNP administration. Both sizes of SiNPs caused local inflammation in the lung and kidney and were detected in the serum and urine at week 13, and the 200-nm particles were also localized to the kidney with no evidence of retention of the 300-nm particles. At week 26, there was some clearance of the 200-nm silica from the kidneys, and urinary levels of SiNPs were reduced but still significant in both 200- and 300 nm-exposed rats. In conclusion, inhaled SiNPs cause chronic kidney injury that progresses despite stopping the SiNP administration. These findings support the hypothesis that human exposure to amorphous silica nanoparticles found in burned sugarcane fields could have a participatory role in chronic kidney disease of unknown etiology.NEW & NOTEWORTHY Inhalation of silica nanoparticles (SiNPs) released during the burning of sugarcane has been postulated to have a role in chronic kidney disease of unknown etiology (CKDu). We administered 200- and 300-nm amorphous SiNPs to rats by aspiration and observed kidney damage with tubular injury and inflammation that persisted even after stopping the SiNP exposure. These findings support the hypothesis that human exposure to SiNPs found in sugarcane ash could have a participatory role CKDu.

A Quantitative Method for Determining Uptake of Silica Nanoparticles in Macrophages by Single Particle Inductively Coupled Plasma-Mass Spectrometry.

Engineered nanomaterials are becoming increasingly ubiquitous in our society, with numerous applications in medicine, consumer products, bioremediation, and advanced materials. As these nanomaterials increase in variety, analyzing their characteristics is of great importance. Single particle inductively coupled plasma-mass spectrometry (SP-ICP-MS) is a high-throughput, sensitive, and robust instrumental analysis method used to simultaneously characterize and quantify nanoparticles in a variety of matrices. One such type of nanoparticle of interest is amorphous silica nanoparticles (SiNPs). SiNPs have widespread use in consumer products such as food and cosmetics and are prime candidates for novel medical applications and uses in environmental bioremediation. Despite their increased use, SiNPs have been shown to have toxicological properties in vitro and in vivo, particularly with regard to the immune system. Because of the potential for increased SiNP exposure in the general public and in occupational settings, examining the relationship that SiNPs have with immune cells such as macrophages to elucidate mechanisms of toxicity is vital. To effectively determine the toxicity of nanoparticles, it is critical to examine dosimetry and the amount of nanoparticles taken up by the cell of interest. Different cell types have different uptake profiles, and varying physicochemical properties govern nanoparticle dosimetry and uptake in cells. Here, we describe a protocol using SP-ICP-MS to quantify and characterize the size, size distribution, and amount of SiNPs present in a cell and medium sample. We use a single-step digestion, which allows for the digestion of biological matrices while simultaneously keeping the SiNPs intact for SP-ICP-MS analysis. Clinically, this approach has the potential to be used as a method for analyzing SiNPs in other biological matrices, potentially as a way of defining SiNP uptake as a biomarker in immune-mediated diseases. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Treatment of cells with silica nanoparticles (SiNPs) and digestion of biological matrices Support Protocol 1: Culturing RAW 264.7 cells for SiNP uptake assay Support Protocol 2: Determination of SiNP size via dynamic light scattering Support Protocol 3: Optimization of sample and ICP-MS parameters for SP-ICP-MS analysis of cells and medium Basic Protocol 2: Analysis and quantification of SiNP uptake in macrophages with SP-ICP-MS.

First-in-class trispecific VHH-Fc based antibody with potent prophylactic and therapeutic efficacy against SARS-CoV-2 and variants.

SARS-CoV-2 and its variants have persisted in this ongoing COVID-19 pandemic. While the vaccines have greatly reduced the COVID-19 cases, hospitalizations, and death, about half of the world remain unvaccinated due to various reasons. Furthermore, the duration of the immunity gained from COVID-19 vaccination is still unclear. Therefore, there is a need for innovative prophylactic and treatment measures. In response to this need, we previously reported on the successful computer-aided development of potent VHH-based multispecific antibodies that were characterized in vitro. Here, we evaluated in vivo efficacy and safety of the lead trispecific VHH-Fc, ABS-VIR-001. Importantly, our data showed that ABS-VIR-001 treatment prevented SARS-CoV-2 infection and death when provided as an intranasal prophylaxis in a humanized ACE-2 mouse model. In addition, ABS-VIR-001 post-exposure treatment was shown to greatly reduce viral loads by as much as 50-fold. A detailed panel of metabolic and cellular parameters demonstrated that ABS-VIR-001 treatment was overall comparable to the PBS treatment, indicating a favorable safety profile. Notably, our inhibition studies show that ABS-VIR-001 continued to demonstrate unwavering efficacy against SARS-CoV-2 mutants, associated with key variants including Delta and Omicron, owing to its multiple epitope design. Lastly, we rigorously tested and confirmed the excellent thermostability of ABS-VIR-001 when heated to 45 °C for up to 4 weeks. Taken together, our study suggests that ABS-VIR-001 is an efficacious and durable prophylaxis and post-exposure treatment for COVID-19 with promising safety and manufacturability features for global distribution.

Chlamydomonas ATX1 is essential for Cu distribution to multiple cupro-enzymes and maintenance of biomass in conditions demanding cupro-enzyme-dependent metabolic pathways.

Copper (Cu) chaperones, of which yeast ATX1 is a prototype, are small proteins with a Cu(I) binding MxCxxC motif and are responsible for directing intracellular Cu toward specific client protein targets that use Cu as a cofactor. The Chlamydomonas reinhardtii ATX1 (CrATX1) was identified by its high sequence similarity with yeast ATX1. Like the yeast homologue, CrATX1 accumulates in iron-deficient cells (but is not impacted by other metal-deficiencies). N- and C-terminally YFP-ATX1 fusion proteins are distributed in the cytoplasm. Reverse genetic analysis using artificial microRNA (amiRNA) to generate lines with reduced CrATX1 abundance and CRISPR/Cpf1 to generate atx1 knockout lines validated a function for ATX1 in iron-poor cells, again reminiscent of yeast ATX1, most likely because of an impact on metalation of the multicopper oxidase FOX1, which is an important component in high-affinity iron uptake. We further identify other candidate ATX1 targets owing to reduced growth of atx1 mutant lines on guanine as a sole nitrogen source, which we attribute to loss of function of UOX1, encoding a urate oxidase, a cupro-enzyme involved in guanine assimilation. An impact of ATX1 on Cu distribution in atx1 mutants is strikingly evident by a reduced amount of intracellular Cu in all conditions probed in this work.

An inherently dangerous fluid warmer design.

A Hotline® fluid warmer is a device commonly used by anesthesia providers in the operating room to warm and infuse blood products and large fluid volumes. The purpose of the fluid warmer is to counter heat loss, which occurs under anesthesia. Despite normal checks performed prior to its use, we discovered a breach in the fluid warming set attached to the Hotline® fluid warmer during blood administration. The breach contaminated the patient's sterile intravenous line. We describe the quality and safety processes we undertook in detail. We discuss the notion that monitoring alarms are an important safety feature of most modern devices utilized by anesthesia providers. We believe the Hotline® fluid warmer lacks a crucial monitor for detecting a breach within the fluid warming set, and therefore recommend the addition of an alarm to improve this device's safety.

The Aryl Hydrocarbon Receptor Modulates Murine Hematopoietic Stem Cell Homeostasis and Influences Lineage-Biased Stem and Progenitor Cells.

The core function of hematopoietic stem and progenitor cells (HSPCs) is to provide lifelong production of all lineages of the blood and immune cells. The mechanisms that modulate HSPC homeostasis and lineage biasing are not fully understood. Growing evidence implicates the aryl hydrocarbon receptor (AHR), an environment-sensing transcription factor, as a regulator of hematopoiesis. AHR ligands modulate the frequency of mature hematopoietic cells in the bone marrow and periphery, while HSPCs from mice lacking AHR (AHR KO) have increased proliferation. Yet, whether AHR modulates HSPC lineage potential and directs differentiation toward specific lineage-biased progenitors is not well understood. This study revealed that AHR KO mice have an increased proportion of myeloid-biased HSCs and myeloid-biased multipotent progenitor (MPP3) cells. Utilizing inducible AHR knockout mice (iAHR KO), it was discovered that acute deletion of AHR doubled the number of MPP3 cells and altered the composition of downstream lineage-committed progenitors, such as increased frequency of pregranulocyte/premonocyte committed progenitors. Furthermore, in vivo antagonism of the AHR led to a 2.5-fold increase in the number of MPP3 cells and promoted myeloid-biased differentiation. Using hematopoietic-specific conditional AHR knockout mice (AHRVav1) revealed that increased frequency of myeloid-biased HSCs and myeloid-biased progenitors is driven by AHR signaling that is intrinsic to the hematopoietic compartment. These findings demonstrate that the AHR plays a pivotal role in regulating steady-state hematopoiesis, influencing HSPC homeostasis and lineage potential. In addition, the data presented provide potential insight into how deliberate modulation of AHR signaling could help with the treatment of a broad range of diseases that require the hematopoietic compartment.

Selective versus nonselective arterial clamping during laparoscopic partial nephrectomy: impact upon renal function in the setting of a solitary kidney in a porcine model.

INTRODUCTION: Laparoscopic partial nephrectomy has emerged as a standard of care for small renal masses. Nevertheless, there remains concern over the potential for irreversible insult to the kidney as a result of exposure to warm ischemia. We aim to investigate the utility of selective segmental arterial clamping as a means to reduce the potential for ischemic damage to a solitary kidney during laparoscopic partial nephrectomy utilizing a porcine model. MATERIALS AND METHODS: A total of 20 domestic swine were randomized into four equal groups. Each subject underwent laparoscopic radical nephrectomy to create the condition of a solitary kidney. On the contralateral side, a laparoscopic lower pole partial nephrectomy was performed, employing either selective or nonselective vascular clamping for either 60 or 90 minutes. Postoperatively, clinical status and serial serum studies were closely monitored for 1 week. RESULTS: There were no intraoperative complications. The 90-minute nonselective clamping produced devastating effects, resulting in rapid deterioration into florid renal failure within 72 hours. The 60-minute nonselective clamping group experienced modest but significant rises in both blood urea nitrogen and creatinine. Both 60- and 90-minute selective clamping groups performed well, with no significant rises in creatinine over a 7-day period, and no instances of renal failure. CONCLUSIONS: Selective arterial clamping is a safe and feasible means of vascular control during laparoscopic partial nephrectomy. In the porcine model, selective clamping appears to improve functional outcomes during prolonged periods of warm ischemic insult. Prospective evaluation of the technique in humans is necessary to determine if selective arterial control confers long-term functional benefits in patients with limited renal reserve.

Open versus laparoscopic retroperitoneal lymph node dissection: assessing adequacy of dissection in a porcine model.

BACKGROUND AND PURPOSE: Laparoscopic retroperitoneal lymph node dissection (L-RPLND) is emerging as a viable alternative to traditional open retroperitoneal lymph node dissection (O-RPLND). Despite numerous reports confirming clinical oncologic equivalency between the two approaches, however, concerns still remain over the adequacy of laparoscopic dissection. We therefore sought to compare the completeness of dissection between O-RPLND and L-RPLND in a porcine model. MATERIALS AND METHODS: Fourteen domestic swine were divided into two equivalent groups. Both groups underwent bilateral retroperitoneal lymph node dissection, approximating templates used in human dissection. In one group, the procedure was performed through an open midline incision, while the other group underwent completely laparoscopic dissection. Tissue was independently analyzed by a pathologist, who recorded lymph node yield based on microscopic evaluation. RESULTS: All animals in the L-RPLND group underwent successful procedures, without the need for conversion. Two open procedures were aborted because of hemorrhage. Mean lymph node yield from O-RPLND was 32, while the mean yield for L-RPLND was 29. This difference was not statistically significant (P=0.65). CONCLUSIONS: In the porcine model, L-RPLND is capable of providing a quality of dissection equivalent to that of O-RPLND, in terms of absolute lymph node yield on microscopic examination. The applicability of this data to human patients, however, may be limited by significant anatomic differences between the human and the pig. Further prospective comparison in human patients is critically needed.

Evaluation of cryoprobe deployment precision with body surface and in situ templates.

OBJECTIVE: To evaluate the precision of cryoprobe targeting with a surface template, an in situ template (on the target organ), or a combined approach. MATERIALS AND METHODS: Fourteen participants placed five 17 G cryoprobes into porcine kidneys in a laparoscopic trainer using a surface template (group 1), an in situ template (group 2) or a combination of the two templates (group 3). The distance from the ideal probe placement was measured both on the anterior and posterior aspect of the kidney. The sequence of attempts was randomized. The distances were compared across the three groups using anova with the adjustment for multiple comparisons. RESULTS: The mean distance from the ideal probe placement was 1.58 cm (anterior) and 1.81 cm (posterior) in group 1, 0.05 cm and 0.39 cm in group 2, and 0.07 cm and 0.22 cm in group 3, respectively. The placement of the probes was significantly more accurate in groups 2 (P < 0.001 anteriorly and P < 0.002 posteriorly) and 3 (P = 0.001 anteriorly and P < 0.001 posteriorly) compared with group 1. There was no significant difference between groups 2 and 3. CONCLUSION: In this in vitro model, the use of internal or combined internal and external templates allows for significantly more precise deployment of 17-G cryoprobes than a standard external template alone.

The many roles of an RNA editor.

The availability of complete genome sequences has made it clear that gene number is not the sole determinant of the complexity of the proteome. Additional complexity that is not readily detected by genome analysis is present in the number and types of RNA transcript that can be derived from each locus. Although alternative splicing is a well-recognized method of generating diversity, the more subtle mechanism of RNA editing is less familiar.