Single-Incision Laparoscopic Appendectomy Using Standard 5 mm Trocars: Single Pediatric Center Experience.

Purpose: Single-incision laparoscopic appendectomy (SILA) for the treatment of appendicitis has been documented. Typically, SILA requires the use of specialized ports, instruments, and materials. The SILA technique at our institution utilizes the same instrumentation as the conventional laparoscopic approach (CLA), thus obviating the need for these specialized products. This study aims to further demonstrate the noninferiority of our SILA technique for the treatment of uncomplicated appendicitis. Materials and Methods: This is a single-institution retrospective review of patients who underwent SILA from 2011 to 2020 to treat uncomplicated appendicitis. Outcomes including demographics, operative time, length of stay (LOS), and common postsurgical complications were evaluated. These SILA cases were matched with up to 3 CLA controls based on age, gender, and weight utilizing the Greedy match method. Patients with an operative diagnosis of perforated appendicitis were excluded. Results: A total of 137 patients underwent SILA at a single institution. A total of 128 patients were in the final cohort after excluding perforated appendicitis. Mean age was 11.9 years. Case-control matching was conducted with 349 controls included. Between cases and controls, SILA had shorter operative time (27.2 minutes versus 43.7 minutes, P < .001) with no difference in mean LOS (42.4 hours versus 42.4 hours, P = .88). There was no difference in complication rate (5.4% versus 8.5%, P = .06). There was no difference in readmission rate (0.8% versus 3.4%, P = .108). Conclusion: These data suggest that for appropriately selected patients, our SILA technique is noninferior to CLA with shortened operative time.

Selective inhibitors of JAK1 targeting an isoform-restricted allosteric cysteine.

The Janus tyrosine kinase (JAK) family of non-receptor tyrosine kinases includes four isoforms (JAK1, JAK2, JAK3, and TYK2) and is responsible for signal transduction downstream of diverse cytokine receptors. JAK inhibitors have emerged as important therapies for immun(onc)ological disorders, but their use is limited by undesirable side effects presumed to arise from poor isoform selectivity, a common challenge for inhibitors targeting the ATP-binding pocket of kinases. Here we describe the chemical proteomic discovery of a druggable allosteric cysteine present in the non-catalytic pseudokinase domain of JAK1 (C817) and TYK2 (C838), but absent from JAK2 or JAK3. Electrophilic compounds selectively engaging this site block JAK1-dependent trans-phosphorylation and cytokine signaling, while appearing to act largely as 'silent' ligands for TYK2. Importantly, the allosteric JAK1 inhibitors do not impair JAK2-dependent cytokine signaling and are inactive in cells expressing a C817A JAK1 mutant. Our findings thus reveal an allosteric approach for inhibiting JAK1 with unprecedented isoform selectivity.

Utilization of Percentage of Predicted Forced Vital Capacity to Stratify Rib Fracture Patients: An Updated Clinical Practice Guideline.

BACKGROUND: Rib fractures are the most common injuries diagnosed after blunt thoracic trauma and are a source of significant morbidity and mortality. Early identification of at-risk patients and initiation of effective analgesia are keys to mitigating complications from these injuries. Multiple tools exist to predict pulmonary decompensation after rib fractures; however, none has found a widespread acceptance. A clinical practice guideline (CPG) utilizing Forced vital capacity (FVC) has been in place at a single institution. The goal of this study is to update the CPG to use percentage of predicted FVC (FVC%) instead of FVC to triage patients with rib fractures. MATERIALS AND METHODS: A retrospective study of 266 patients with rib fractures was conducted. Patients were divided into 3 groups based on FVC of <1000 mL, 1001-1500 mL, or >1500 mL for analysis. Data were analyzed with analysis of variance, and Youden's J Index was used to identify inflection points. RESULTS: Patients in the high-risk category were more likely to be women, older than 65 years, admitted to the intensive care unit (ICU), transferred to the ICU, require intubation, and have overall longer hospital and ICU stays. The updated CPG triage cutoffs for admission to ICU, stepdown, and floor were redefined as FVC% values of <25%, 25-45%, and >45%, respectively. DISCUSSION: The updated CPG using FVC% may more accurately identify patients with compromised physiology and be a better tool to help predict patients who are at risk for decompensation following rib fractures. A validation study for the updated CPG is in progress.

DCAF11 Supports Targeted Protein Degradation by Electrophilic Proteolysis-Targeting Chimeras.

Ligand-induced protein degradation has emerged as a compelling approach to promote the targeted elimination of proteins from cells by directing these proteins to the ubiquitin-proteasome machinery. So far, only a limited number of E3 ligases have been found to support ligand-induced protein degradation, reflecting a dearth of E3-binding compounds for proteolysis-targeting chimera (PROTAC) design. Here, we describe a functional screening strategy performed with a focused library of candidate electrophilic PROTACs to discover bifunctional compounds that degrade proteins in human cells by covalently engaging E3 ligases. Mechanistic studies revealed that the electrophilic PROTACs act through modifying specific cysteines in DCAF11, a poorly characterized E3 ligase substrate adaptor. We further show that DCAF11-directed electrophilic PROTACs can degrade multiple endogenous proteins, including FBKP12 and the androgen receptor, in human prostate cancer cells. Our findings designate DCAF11 as an E3 ligase capable of supporting ligand-induced protein degradation via electrophilic PROTACs.

Fine needle biopsy of malignant tumors of the liver: a retrospective study of 624 cases from a single institution experience.

BACKGROUND: Liver is one of the most common organs involved by metastatic neoplasms. In addition, a number of primary tumors can arise in the liver. Fine needle biopsy (FNB) is the most commonly used method for diagnosis of liver masses. Not much literature is available during the past 10 years about FNB of liver tumors. All large studies were performed more than 15 years ago. With the introduction of new disease entities, new tumor classification systems, and new diagnostic methods, updated documentation of FNB of liver neoplasms is much needed. METHODS: Liver FNB cases that were diagnosed as "Positive for Malignancy" between 2010 and 2018 were retrieved from the cytopathology database in our institution. Patient medical records, cytopathology and surgical pathology reports, and slides from selected cases were retrieved and reviewed. RESULTS: Over 30 different types of malignant tumors were identified in 624 malignant FNB cases, with the most common tumors being metastatic colorectal and pancreatic adenocarcinomas. Rare tumors include EBV-positive leiomyosarcoma, mesothelioma, and paraganglioma, among others. A subset of patients presented with widespread metastases involving liver with no known history. Identifying the primary sites in those cases can be challenging. We also found that in our practice, a significant number of hepatocellular carcinoma were diagnosed by FNB in recent years. CONCLUSIONS: A tremendous variety of neoplasms can occur in liver. Accurate diagnosis is essential for proper patient management. Familiarization with morphological features and judicious usage of ancillary studies are essential for accurate diagnosis.

Protocol driven management of suspected common duct stones: A Southwestern Surgical Congress multi-centered trial.

BACKGROUND: Several options exist for the diagnosis and management of suspected common duct stones. We hypothesized that a protocol-directed approach would shorten length of stay in this patient population. METHODS: Patients from four participating institutions with a peak bilirubin <4 mg/dL underwent surgery as the initial procedure, whereas patients with a bilirubin ≥4 mg/dL underwent endoscopy. The primary endpoint was length of stay. Analysis involved chi square and Wilcoxon-Mann-Whitney test with significance at p < 0.05. RESULTS: 214 patients were managed under the protocol during six-month study period. 111 patients (52%) required endoscopy and surgery. Length of stay and the number of MRCPs performed pre-operatively significantly decreased following protocol implementation (p < 0.05). CONCLUSIONS: "Surgery first" approach in patients with bilirubin <4 ml/dL resulted in low morbidity and mortality, reduced MRCP, and length of stay.

Overutilization of Test for Hemoglobinopathy Evaluation: Experience from a Tertiary Care Academic Medical Center.

BACKGROUND: Hemoglobin electrophoresis is a common clinical laboratory test for the identification of hemoglobinopathies in clinical practice. We investigated the utilization of this test in our academic teaching hospital and hypothesized that hemoglobin electrophoresis evaluation is overutilized at this institution. METHODS: 128 consecutive cases were analyzed and their medical records were studied to determine the clinical indication for the hemoglobin electrophoresis. RESULTS: Of the 128 cases studied, only 44% of cases had a justifiable reason for obtaining the hemoglobin electrophoresis, whereas the remaining 56% of cases did not have an acceptable indication for obtaining the test. CONCLUSIONS: We conclude that hemoglobin electrophoresis is overutilized in our academic medical center and we recommend consultation with clinical pathologists prior to ordering such tests.

Biotin at High Concentration Interferes with the LOCI Digoxin Assay but the PETINIA Phenytoin Assay Is Not Affected.

Many automated immunoassays incorporate biotinylated antibodies and streptavidin-coated magnetic beads in the assay design. Biotin at elevated concentrations may interfere with these immunoassays. We evaluated potential interference of biotin on serum digoxin (LOCI assay utilizing biotinylated antibody) and phenytoin (PETINIA assay; no biotinylated antibody) measurements using the Vista 1500 analyzer. Aliquots of drug-free serum pool were supplemented with various biotin concentrations (range: 1 ng/mL to 250 ng/mL) followed by measuring apparent digoxin and phenytoin levels using appropriate immunoassays. In the second set of experiments, one serum pool was prepared from patients taking digoxin and another from patients taking phenytoin. Then aliquots of these serum pools were further supplemented with biotin followed by measuring digoxin or phenytoin concentrations. We observed apparent digoxin levels at 50 ng/mL biotin concentration or higher and also significant interference of biotin in serum digoxin measurement at a biotin concentration of 250 ng/mL. In contrast, we observed no interference of biotin in serum phenytoin measurement. We conclude that biotin interferes with the LOCI digoxin assay at a high concentration only.

The Conserved RNA Exonuclease Rexo5 Is Required for 3' End Maturation of 28S rRNA, 5S rRNA, and snoRNAs.

Non-coding RNA biogenesis in higher eukaryotes has not been fully characterized. Here, we studied the Drosophila melanogaster Rexo5 (CG8368) protein, a metazoan-specific member of the DEDDh 3'-5' single-stranded RNA exonucleases, by genetic, biochemical, and RNA-sequencing approaches. Rexo5 is required for small nucleolar RNA (snoRNA) and rRNA biogenesis and is essential in D. melanogaster. Loss-of-function mutants accumulate improperly 3' end-trimmed 28S rRNA, 5S rRNA, and snoRNA precursors in vivo. Rexo5 is ubiquitously expressed at low levels in somatic metazoan cells but extremely elevated in male and female germ cells. Loss of Rexo5 leads to increased nucleolar size, genomic instability, defective ribosome subunit export, and larval death. Loss of germline expression compromises gonadal growth and meiotic entry during germline development.

Multimodal Genetic Approach for Molecular Imaging of Vasculature in a Mouse Model of Melanoma.

PURPOSE: In this study, we evaluated a genetic approach for in vivo multimodal molecular imaging of vasculature in a mouse model of melanoma. PROCEDURES: We used a novel transgenic mouse, Ts-Biotag, that genetically biotinylates vascular endothelial cells. After inoculating these mice with B16 melanoma cells, we selectively targeted endothelial cells with (strept)avidinated contrast agents to achieve multimodal contrast enhancement of Tie2-expressing blood vessels during tumor progression. RESULTS: This genetic targeting system provided selective labeling of tumor vasculature and showed in vivo binding of avidinated probes with high specificity and sensitivity using microscopy, near infrared, ultrasound, and magnetic resonance imaging. We further demonstrated the feasibility of conducting longitudinal three-dimensional (3D) targeted imaging studies to dynamically assess changes in vascular Tie2 from early to advanced tumor stages. CONCLUSIONS: Our results validated the Ts-Biotag mouse as a multimodal targeted imaging system with the potential to provide spatio-temporal information about dynamic changes in vasculature during tumor progression.

The contribution of mutant GBA to the development of Parkinson disease in Drosophila.

Gaucher disease (GD) results from mutations in the acid ß-glucocerebrosidase (GCase) encoding gene, GBA, which leads to accumulation of glucosylceramides. GD patients and carriers of GD mutations have a significantly higher propensity to develop Parkinson disease (PD) in comparison to the non-GD population. In this study, we used the fruit fly Drosophila melanogaster to show that development of PD in carriers of GD mutations results from the presence of mutant GBA alleles. Drosophila has two GBA orthologs (CG31148 and CG31414), each of which has a minos insertion, which creates C-terminal deletion in the encoded GCase. Flies double heterozygous for the endogenous mutant GBA orthologs presented Unfolded Protein Response (UPR) and developed parkinsonian signs, manifested by death of dopaminergic cells, defective locomotion and a shorter life span. We also established transgenic flies carrying the mutant human N370S, L444P and the 84GG variants. UPR activation and development of parkinsonian signs could be recapitulated in flies expressing these three mutant variants.UPR and parkinsonian signs could be partially rescued by growing the double heterozygous flies, or flies expressing the N370S or the L444P human mutant GCase variants, in the presence of the pharmacological chaperone ambroxol, which binds and removes mutant GCase from the endoplasmic reticulum (ER). However flies expressing the 84GG mutant, that does not express mature GCase, did not exhibit rescue by ambroxol. Our results strongly suggest that the presence of a mutant GBA allele in dopaminergic cells leads to ER stress and to their death, and contributes to development of PD.

Engineering an effective Mn-binding MRI reporter protein by subcellular targeting.

PURPOSE: Manganese (Mn) is an effective contrast agent and biologically active metal, which has been widely used for Mn-enhanced MRI (MEMRI). The purpose of this study was to develop and test a Mn binding protein for use as a genetic reporter for MEMRI. METHODS: The bacterial Mn-binding protein, MntR was identified as a candidate reporter protein. MntR was engineered for expression in mammalian cells, and targeted to different subcellular organelles, including the Golgi Apparatus where cellular Mn is enriched. Transfected HEK293 cells and B16 melanoma cells were tested in vitro and in vivo, using immunocytochemistry, MR imaging and relaxometry. RESULTS: Subcellular targeting of MntR to the cytosol, endoplasmic reticulum and Golgi apparatus was verified with immunocytochemistry. After targeting to the Golgi, MntR expression produced robust R1 changes and T1 contrast in cells, in vitro and in vivo. Co-expression with the divalent metal transporter DMT1, a previously described Mn-based reporter, further enhanced contrast in B16 cells in culture, but in the in vivo B16 tumor model tested was not significantly better than MntR alone. CONCLUSION: This second-generation reporter system both expands the capabilities of genetically encoded reporters for imaging with MEMRI and provides important insights into the mechanisms of Mn biology which create endogenous MEMRI contrast.

Sept4/ARTS regulates stem cell apoptosis and skin regeneration.

Adult stem cells are essential for tissue homeostasis and wound repair. Their proliferative capacity must be tightly regulated to prevent the emergence of unwanted and potentially dangerous cells, such as cancer cells. We found that mice deficient for the proapoptotic Sept4/ARTS gene have elevated numbers of hair follicle stem cells (HFSCs) that are protected against apoptosis. Sept4/ARTS(-/-) mice display marked improvement in wound healing and regeneration of hair follicles. These phenotypes depend on HFSCs, as indicated by lineage tracing. Inactivation of XIAP, a direct target of ARTS, abrogated these phenotypes and impaired wound healing. Our results indicate that apoptosis plays an important role in regulating stem cell-dependent regeneration and suggest that this pathway may be a target for regenerative medicine.

Divalent metal transporter, DMT1: a novel MRI reporter protein.

Manganese (Mn)-enhanced MRI (MEMRI) has found a growing number of applications in anatomical and functional imaging in small animals, based on the cellular uptake of Mn ions in the brain, heart, and other organs. Previous studies have relied on endogenous mechanisms of paramagnetic Mn ion uptake and enhancement. To genetically control MEMRI signals, we reverse engineered a major component of the molecular machinery involved in Mn uptake, the divalent metal transporter, DMT1. DMT1 provides positive cellular enhancement in a manner that is highly sensitive and dynamic, allowing greater spatial and temporal resolution for MRI compared to previously proposed MRI reporters such as ferritin. We characterized the MEMRI signal enhancement properties of DMT1-expressing cells, both in vitro and in vivo in mouse models of cancer and brain development. Our results show that DMT1 provides an effective genetic MRI reporter for a wide range of biological and preclinical imaging applications.

Novel genetic approach for in vivo vascular imaging in mice.

RATIONALE: The formation and maintenance of a functional vasculature is essential for normal embryonic development, and genetic changes that affect the vasculature underlie pathogenesis in many human diseases. In vivo imaging in mouse models is required to understand the full complexity of mammalian vascular formation, which is a dynamic and 3-dimensional process. Optical microscopy of genetically expressed fluorescent reporter proteins offers high resolution but limited depth of penetration in vivo. Conversely, there are a plethora of molecular probes for alternative in vivo vascular imaging modalities, but few options for genetic control of contrast enhancement. OBJECTIVE: To develop a reporter system for multimodal imaging of genetic processes involved in mammalian vascular biology. METHODS AND RESULTS: To approach this problem, we developed an optimal tagging system based on Biotag-BirA technology. In the resulting Biotag reporter system, coexpression of 2 interacting proteins results in biotin labeling of cell membranes, thus enabling multimodal imaging with "avidinated" probes. To assess this approach for in vivo imaging, we generated transgenic mice that expressed the Biotag-BirA transgene from a minimal Tie2 promoter. A variety of imaging methods were used to show the utility of this approach for quantitative analysis in embryonic and adult models of vascular development, using intravascular injection of avidinated probes for near infrared, ultrasound, and magnetic resonance imaging. CONCLUSIONS: The present results demonstrate the versatility of the Biotag system for studies of vascular biology in genetically engineered mice, providing a robust approach for multimodal in vivo imaging of genetic processes in the vasculature.

In Vitro and In Vivo studies of monoclonal antibodies with prominent bactericidal activity against Burkholderia pseudomallei and Burkholderia mallei.

Our laboratory has developed more than a hundred mouse monoclonal antibodies (MAbs) against Burkholderia pseudomallei and Burkholderia mallei. These antibodies have been categorized into different groups based on their specificities and the biochemical natures of their target antigens. The current study first examined the bactericidal activities of a number of these MAbs by an in vitro opsonic assay. Then, the in vivo protective efficacy of selected MAbs was evaluated using BALB/c mice challenged intranasally with a lethal dose of the bacteria. The opsonic assay using dimethyl sulfoxide-treated human HL-60 cells as phagocytes revealed that 19 out of 47 tested MAbs (40%) have prominent bactericidal activities against B. pseudomallei and/or B. mallei. Interestingly, all MAbs with strong opsonic activities are those with specificity against either the capsular polysaccharides (PS) or the lipopolysaccharides (LPS) of the bacteria. On the other hand, none of the MAbs reacting to bacterial proteins or glycoproteins showed prominent bactericidal activity. Further study revealed that the antigenic epitopes on either the capsular PS or LPS molecules were readily available for binding in intact bacteria, while the epitopes on proteins/glycoproteins were less accessible to the MAbs. Our in vivo study showed that four MAbs reactive to either the capsular PS or LPS were highly effective in protecting mice against lethal bacterial challenge. The result is compatible with that of our in vitro study. The MAbs with the highest protective efficacy are those reactive to either the capsular PS or LPS of the Burkholderia bacteria.

In vivo MRI of neural cell migration dynamics in the mouse brain.

Multipotent neuroblasts (NBs) are produced throughout life by neural stem cells in the forebrain subventricular zone (SVZ), and are able to travel long distances to the olfactory bulb. On arrival in the bulb, migrating NBs normally replace olfactory neurons, raising interest in their potential for novel cell replacement therapies in various disease conditions. An understanding of the migratory capabilities of NBs is therefore important, but as yet quantitative in vivo measurement of cell migration has not been possible. In this study, targeted intracerebral injections of iron-oxide particles to the mouse SVZ were used to label resident NBs in situ, and their migration was tracked noninvasively over time with magnetic resonance imaging (MRI). Quantitative intensity metrics were employed to identify labeled cells and to show that cells are able to travel at speeds up to 100 microm/h en route to the olfactory bulb, but that distribution through the olfactory bulb occurs at a much slower rate. In addition, comparison of histological and MRI measures of iron-oxide particle distribution were in excellent agreement. Immunohistochemistry analysis 1-3 weeks after labeling revealed that the majority of labeled cells in the olfactory bulb were immature neurons, although iron-oxide particles were also found in astrocytes and microglia. This work indicates that dynamic measurements of endogenous cell migration can be made with MRI and represents the first in vivo measurement of NB migration rates. The use of MRI in future studies tracking endogenous NB cells will permit a more complete evaluation of their role during homeostasis at various developmental stages and during disease progression.