KaryoCreate: A CRISPR-based technology to study chromosome-specific aneuploidy by targeting human centromeres.

Aneuploidy, the presence of chromosome gains or losses, is a hallmark of cancer. Here, we describe KaryoCreate (karyotype CRISPR-engineered aneuploidy technology), a system that enables the generation of chromosome-specific aneuploidies by co-expression of an sgRNA targeting chromosome-specific CENPA-binding ɑ-satellite repeats together with dCas9 fused to mutant KNL1. We design unique and highly specific sgRNAs for 19 of the 24 chromosomes. Expression of these constructs leads to missegregation and induction of gains or losses of the targeted chromosome in cellular progeny, with an average efficiency of 8% for gains and 12% for losses (up to 20%) validated across 10 chromosomes. Using KaryoCreate in colon epithelial cells, we show that chromosome 18q loss, frequent in gastrointestinal cancers, promotes resistance to TGF-ß, likely due to synergistic hemizygous deletion of multiple genes. Altogether, we describe an innovative technology to create and study chromosome missegregation and aneuploidy in the context of cancer and beyond.

SIRT6 Is Responsible for More Efficient DNA Double-Strand Break Repair in Long-Lived Species.

DNA repair has been hypothesized to be a longevity determinant, but the evidence for it is based largely on accelerated aging phenotypes of DNA repair mutants. Here, using a panel of 18 rodent species with diverse lifespans, we show that more robust DNA double-strand break (DSB) repair, but not nucleotide excision repair (NER), coevolves with longevity. Evolution of NER, unlike DSB, is shaped primarily by sunlight exposure. We further show that the capacity of the SIRT6 protein to promote DSB repair accounts for a major part of the variation in DSB repair efficacy between short- and long-lived species. We dissected the molecular differences between a weak (mouse) and a strong (beaver) SIRT6 protein and identified five amino acid residues that are fully responsible for their differential activities. Our findings demonstrate that DSB repair and SIRT6 have been optimized during the evolution of longevity, which provides new targets for anti-aging interventions.

Practice Patterns in Utilization of Atherectomy and Embolic Protection Devices in Inpatient and Outpatient Treatment Settings.

OBJECTIVES: The frequency of atherectomy in lower extremity arterial disease has increased substantially over the past several years, specifically in the office-based laboratory (OBL) setting, yet the efficacy compared to other interventions and the consequences of distal embolization remain unknown. Embolic Protection Devices (EPD) have been used at varying rates depending on physician and practice setting. Previous studies have described lesion characteristics to consider when weighing the benefits and drawbacks associated with device usage. Our study focuses on the use of atherectomy and EPD in femoropopliteal arterial disease to better characterize resource usage trends and postoperative outcomes in the inpatient and OBL interventional settings. METHODS: We conducted a retrospective analysis on endovascular interventions performed for femoral-popliteal occlusive disease that were entered into the Vascular Quality Initiative (VQI) data registry between 2017-2021. A 1:1 greedy-match, adjusted analysis based on inpatient or OBL location of procedure was utilized to compare the groups. Hierarchical logistical regression with selective use of principal component analysis was utilized to further explore the differences in EPD usage and immediate postoperative outcomes. A proportional hazard model was used to demonstrate differences in reintervention rates up to two years postoperatively between patients who underwent atherectomy in the inpatient vs OBL treatment setting. RESULTS: 2,849 matched pairs were included in the final analysis. In our cohort, there was 22% EPD usage overall, 40% in the hospital setting and 4.4% in the OBL setting (p<0.001). Among the patients with available follow-up information, OBL intervention setting increased probability of reintervention by 18% at 2 years postoperatively compared to the inpatient setting, however there was no difference associated with EPD placement and rate of reintervention. CONCLUSIONS: Use of EPD in the OBL setting compared to the hospital setting is dramatically decreased, however, no increased incidence of postoperative complications was seen compared to procedures performed in the hospital setting when controlling for patient and lesion characteristics. Patients with available follow-up data were more likely to undergo ipsilateral reintervention between 6 months and 2 years postoperatively if atherectomy was done in the OBL setting. Dedicated studies are encouraged to ensure patient safety, effective resource allocation, and long-term efficacy of OBL atherectomy as an ever-growing number of peripheral arterial procedures are transitioned to the OBL setting.

A novel approach to saline/contrast delivery in excimer laser coronary atherectomy (ELCA) to enhance efficacy: MAXCon ELCA technique.

The advent of excimer laser coronary atherectomy (ELCA) nearly four decades ago heralded a novel way to treat complex lesions, both coronary and peripheral, which were previously untraversable and thus untreatable. These complex lesions include heavily calcified lesions, ostial lesions, bifurcation lesions, chronic total occlusions, in-stent restenosis (including stent underexpansion), and degenerative saphenous vein grafts. We discuss the technology of ELCA, its indications, applications, and complications, and suggest the "MAXCon ELCA" technique for better outcomes without increased risk. Lastly, we present a case of MAXCon ELCA effectively treating a complex lesion.

Unexpected tobacco etch virus (TEV) protease cleavage of recombinant human proteins.

The tobacco etch virus (TEV) protease is a commonly used reagent for removal of solubility and purification tags from recombinant proteins and is cited as being highly specific for its canonical cleavage site. Flexibility in some amino acids within this recognition sequence has been described in the literature but researchers generally assume few native human proteins will carry off-target sequences for TEV cleavage. We report here the aberrant cleavage of three human proteins with non-canonical TEV protease cleavage sites and identify broader sequence specificity rules that can be used to predict unwanted cleavage of recombinant proteins. Using these rules, 456 human proteins were identified that could be substrates for unwanted TEV protease cleavage.

Molecular Mechanisms of Vascular Health: Insights From Vascular Aging and Calcification.

Cardiovascular disease is the most common cause of death worldwide, especially beyond the age of 65 years, with the vast majority of morbidity and mortality due to myocardial infarction and stroke. Vascular pathology stems from a combination of genetic risk, environmental factors, and the biologic changes associated with aging. The pathogenesis underlying the development of vascular aging, and vascular calcification with aging, in particular, is still not fully understood. Accumulating data suggests that genetic risk, likely compounded by epigenetic modifications, environmental factors, including diabetes and chronic kidney disease, and the plasticity of vascular smooth muscle cells to acquire an osteogenic phenotype are major determinants of age-associated vascular calcification. Understanding the molecular mechanisms underlying genetic and modifiable risk factors in regulating age-associated vascular pathology may inspire strategies to promote healthy vascular aging. This article summarizes current knowledge of concepts and mechanisms of age-associated vascular disease, with an emphasis on vascular calcification.

Retrospective evaluation of MRI findings in arthroscopically confirmed cases of hypermobile lateral meniscus.

OBJECTIVE: To identify preoperative MRI findings in patients with arthroscopically confirmed hypermobile lateral meniscus utilizing a standard MRI knee protocol, with comparison to normal control and lateral meniscal tear groups. SUBJECTS AND METHODS: All patients with arthroscopically confirmed hypermobile lateral meniscus diagnosed at our institution were retrospectively identified. The following structures were evaluated on preoperative knee MRIs: superior and inferior popliteomeniscal fascicles, lateral meniscus and meniscocapsular junction, popliteal hiatus, and soft tissue edema around the popliteal hiatus. The same MRI features were evaluated in the normal control and lateral meniscal tear groups. RESULTS: Study, normal control, and lateral meniscal tear patients (18 each) were included. In the study group, 94.4% had superior popliteomeniscal fascicle abnormality, 89.0% had inferior popliteomeniscal fascicle abnormality, and 72.2% had lateral meniscal abnormality. Incidence of these abnormalities was significantly higher than in the normal control group. Meniscal abnormalities in the study group all involved the posterior horn meniscocapsular junction, 12/13 of which had vertical signal abnormality at the junction and 1/13 with anterior subluxation of the entire posterior horn. Popliteus hiatus measurements were largest in the lateral meniscal tear group. CONCLUSION: In patients with hypermobile lateral meniscus, the combination of popliteomeniscal fascicle abnormality and vertical signal abnormality at the meniscocapsular junction was seen in the majority of patients. Popliteomeniscal fascicle signal abnormality without identifiable lateral meniscal injury was the next most common imaging appearance. Radiologists may provide valuable information by suggesting the diagnosis of hypermobile lateral meniscus in such cases.

Is There Utility in Preoperative Testing of Hemoglobin Before Primary Cheiloplasty?

OBJECTIVE: To examine whether a preoperative hemoglobin of less than 10 g/dL is associated with a higher rate of perioperative complications. DESIGN: Retrospective review. SETTING: Tertiary academic hospital at Arkansas Children's Hospital of Little Rock, Arkansas. PATIENTS: A retrospective chart review evaluated patients undergoing primary cleft lip surgery from 2012 to 2017. INTERVENTIONS: No prospective intervention was performed for this study care. MAIN OUTCOME MEASURES: Age, sex, medical history, weight, and perioperative complications. Hemoglobin level was collected in the preoperative area. The primary outcome was rate of perioperative complications including infection, dehiscence, return to the operating room, unplanned admission, and emergency department visit within two weeks postoperatively. RESULTS: 105 patients undergoing primary cheiloplasty met inclusion criteria. Hemoglobin levels were obtained on all patients. 93.3% (n = 98) of patients had a hemoglobin of >10 g/dL before surgery, and 6.6% (n = 7) had levels <10 g/dL. 1 of 7 patients with a hemoglobin of <10 g/dL experienced a postoperative complication (Tet spell) and one patient with a hemoglobin of >10 g/dL experienced a postoperative complication (unplanned intensive care admission for respiratory distress). CONCLUSIONS: Post-operative complications are rare after primary cheiloplasty in patients with low or normal hemoglobin levels. The results of this study show that a preoperative hemoglobin of <10 g/dL does not predict perioperative complications in patients undergoing primary cheiloplasty.

Gluteus Maximus Distal Myotendinous Junction Tear in a Pickleball Player: A Case Report.

ABSTRACT: A 72-year-old male presented for evaluation of a 2-wk history left buttock pain that began while playing pickleball. He sustained a left inversion ankle sprain while in a squatted position and landed on his left buttock. Four days after his injury, he developed extensive bruising involving his lower back, buttock, and left thigh. On examination, he had tenderness to palpation at the left side of the sacrum and in the region of the deep external rotators. Left hip range of motion was full in extension but limited to 90° of flexion, which reproduced left-sided buttock pain. External rotation provoked pain, but internal rotation was full and pain free. MRI of the pelvis demonstrated a grade 2 partial thickness tear of the left gluteus maximus muscle at its distal myotendinous junction with associated retraction and intramuscular hematoma. He was managed with compression with biking shorts, icing, acetaminophen, and physical therapy. He returned to pickleball approximately 4 wk after his injury, and at his 4-wk follow-up, he reported 99% improvement in his symptoms with the only remaining complaint being minimal discomfort with gluteal stretching.

Unique Evolution of Antiviral Tetherin in Bats.

Bats are recognized as important reservoirs of viruses deadly to other mammals, including humans. These infections are typically nonpathogenic in bats, raising questions about host response differences that might exist between bats and other mammals. Tetherin is a restriction factor which inhibits the release of a diverse range of viruses from host cells, including retroviruses, coronaviruses, filoviruses, and paramyxoviruses, some of which are deadly to humans and transmitted by bats. Here, we characterize the tetherin genes from 27 bat species, revealing that they have evolved under strong selective pressure, and that fruit bats and vesper bats express unique structural variants of the tetherin protein. Tetherin was widely and variably expressed across fruit bat tissue types and upregulated in spleen tissue when stimulated with Toll-like receptor agonists. The expression of two computationally predicted splice isoforms of fruit bat tetherin was verified. We identified an additional third unique splice isoform which includes a C-terminal region that is not homologous to known mammalian tetherin variants but was functionally capable of restricting the release of filoviral virus-like particles. We also report that vesper bats possess and express at least five tetherin genes, including structural variants, more than any other mammal reported to date. These findings support the hypothesis of differential antiviral gene evolution in bats relative to other mammals. IMPORTANCE Bats are an important host of various viruses which are deadly to humans and other mammals but do not cause outward signs of illness in bats. Furthering our understanding of the unique features of the immune system of bats will shed light on how they tolerate viral infections, potentially informing novel antiviral strategies in humans and other animals. This study examines the antiviral protein tetherin, which prevents viral particles from escaping their host cell. Analysis of tetherin from 27 bat species reveals that it is under strong evolutionary pressure, and we show that multiple bat species have evolved to possess more tetherin genes than other mammals, some of which encode structurally unique tetherins capable of activity against different viral particles. These data suggest that bat tetherin plays a potentially broad and important role in the management of viral infections in bats.

Discovery of arylsulfonamides as a novel class of allosteric integrase inhibitors with antiviral activity.

Lens epithelial-derived growth factor (LEDGF) increases the efficiency of proviral DNA integration into the host genome by interacting with HIV integrase (IN) and directing it to a chromatin environment that favors viral transcription. Allosteric integrase inhibitors (ALLINIs), such as known 2-(tert-butoxy)acetic acid (1), bind to the LEDGF pocket on the catalytic core domain (CCD) of IN, but exert more potent antiviral activities by inhibition of late-stage HIV-1 replication events than through disruption of proviral integration at an earlier phase. A high-throughput screen (HTS) for compounds that disrupt IN-LEDGF interaction led to the identification of a novel arylsulfonamide series, as exemplified by 2, possessing ALLINI-like properties. Further SAR studies led to more potent compound 21 and provided key chemical biology probes revealing that arylsulfonamides are a novel class of ALLINIs with a distinct binding mode than that of 2-(tert-butoxy)acetic acids.

Filling a nick in NIK: Extending the half-life of a NIK inhibitor through structure-based drug design.

Inhibition of NF-κB inducing kinase (NIK) has been pursued as a promising therapeutic target for autoimmune disorders due to its highly regulated role in key steps of the NF-κB signaling pathway. Previously reported NIK inhibitors from our group were shown to be potent, selective, and efficacious, but had higher human dose projections than desirable for immunology indications. Herein we report the clearance-driven optimization of a NIK inhibitor guided by metabolite identification studies and structure-based drug design. This led to the identification of an azabicyclo[3.1.0]hexanone motif that attenuated in vitro and in vivo clearance while maintaining NIK potency and increasing selectivity over other kinases, resulting in a greater than ten-fold reduction in predicted human dose.

Infectious KoRV-related retroviruses circulating in Australian bats.

Bats are reservoirs of emerging viruses that are highly pathogenic to other mammals, including humans. Despite the diversity and abundance of bat viruses, to date they have not been shown to harbor exogenous retroviruses. Here we report the discovery and characterization of a group of koala retrovirus-related (KoRV-related) gammaretroviruses in Australian and Asian bats. These include the Hervey pteropid gammaretrovirus (HPG), identified in the scat of the Australian black flying fox (Pteropus alecto), which is the first reproduction-competent retrovirus found in bats. HPG is a close relative of KoRV and the gibbon ape leukemia virus (GALV), with virion morphology and Mn2+-dependent virion-associated reverse transcriptase activity typical of a gammaretrovirus. In vitro, HPG is capable of infecting bat and human cells, but not mouse cells, and displays a similar pattern of cell tropism as KoRV-A and GALV. Population studies reveal the presence of HPG and KoRV-related sequences in several locations across northeast Australia, as well as serologic evidence for HPG in multiple pteropid bat species, while phylogenetic analysis places these bat viruses as the basal group within the KoRV-related retroviruses. Taken together, these results reveal bats to be important reservoirs of exogenous KoRV-related gammaretroviruses.

Outcomes After Pharyngeal Flap Surgery in Children: A Comparison of Lined Versus Unlined Flaps.

OBJECTIVE: The addition of a uvular flap (PFU) was hypothesized to improve outcomes over standard pharyngeal flap (PF) for correction of velopharyngeal dysfunction. We report differences in outcomes of PF vs PFU at our institution. DESIGN: Retrospective cohort study. SETTING: Tertiary children's hospital. PATIENTS: Children who underwent PF or PFU with the three highest-volume surgeons at our institution in 2004-2017. OUTCOME MEASURES: We examined differences in complications between groups, frequency and type of revision surgery, and speech-related measures including nasometry, pressure-flow testing (PFT) and perceptual speech analysis (PSA). RESULTS: 160 patients were included, 41 PF and 119 PFU (including 18 with Hogan technique). Patients undergoing PFU were older (7.6 yr vs 6.0 yr; p = 0.037) and more likely to have cleft palate (63/119 vs 14/41; p = 0.047). There was no significant difference in complications. With PFU, a decrease in airspace contracting revision surgeries was noted, (4/119 vs 8/41; p = 0.002) which drove a reduction in revision surgery of all types (7/119 vs 13/41; p = 0.033). However, patients that did undergo revision surgery after PFU underwent more revision procedures (p = 0.032). PSA scores were found to be lower (less hypernasal) after PFU (p = 0.009) compared to PF. Objective speech measures had varying results, with nasometry demonstrating a significant difference between groups (p = 0.001), while PFT (p = 0.525) did not demonstrate a statistical difference. CONCLUSION: The use of a uvular lining flap in pharyngeal flap surgery may be associated with improved long term surgical outcomes, including both improvements in subjective and objective testing and a lower rate of revision surgery, without increased complications.

Ergonomics for Cleft Providers: A Brief Communication.

Musculoskeletal (MSK) injuries are the most common and debilitating work-related injuries among healthcare providers. These injuries often occur due to a lack of awareness and insufficient guidance during the early years of medical training. Recognizing the need to address this issue, the Comprehensive Cleft Care Workshop (CCCW) has taken steps to integrate an ergonomics session into its curriculum. The goal of this initiative is to enhance awareness on ergonomics, improve the integration of ergonomics into daily routine, and ultimately reduce the occurrence of MSK injuries among healthcare professionals.

Single-Stage Posterior Vomerine Ostectomy and Primary Cheiloplasty in Patients with Bilateral Cleft Lip & Palate and Protuberant Premaxilla.

OBJECTIVE: A protruded premaxilla has always been challenging to care for by cleft care professionals. This study aims to fortify the use of a single-stage premaxillary setback, with posterior vomerine ostectomy and primary cheiloplasty to achieve proper care for patients with bilateral cleft lip and palate (BCLP) and protruded premaxilla. DESIGN: Longitudinal retrospective analysis. SETTING: Twenty-three outreach programs to four countries (Ecuador, Lebanon, Peru, and El-Salvador) between 2016-2022. PATIENTS/PARTICIPANTS: Sixty-five patients between the ages of 3 months and 6 years and 5 months, with BCLP and severely protruded premaxilla underwent premaxillary setback via posterior vomerine ostectomy and primary cheiloplasty. Patients with diagnosed syndromes and inaccessible vomer bone due to fused palates were excluded from the study. INTERVENTIONS: Premaxillary setback with posterior vomerine ostectomy, bilateral gingivoperiosteoplasties (GPP), and primary cheiloplasty. MAIN OUTCOME MEASURE(S): Postoperative complications and aesthetic outcomes. RESULTS: The mean age at surgery was 13.17 ± 14.1 months, with an average follow-up time of 26 ± 17 months. Patients underwent their procedures in Ecuador (72%), Peru (9%), Lebanon (8%) and El-Salvador (1%). The majority of patients were aged 1 year or less (66.7%) and were males (58.5%). All patients were operated on successfully and had good aesthetic outcomes. Only one patient developed partial necrosis. CONCLUSION: Patients with BCLP and severe premaxillary protrusion have always carried immense social, psychological, and financial burdens, especially in outreach settings. Our described single-stage technique has proven to be safe and effective with good aesthetic results. Further follow-up after primary repair should be done to document and ensure proper facial growth and normal nasolabial maturation.

The First Hybrid International Educational Comprehensive Cleft Care Workshop.

OBJECTIVE: Describe the first hybrid global simulation-based comprehensive cleft care workshop, evaluate impact on participants, and compare experiences based on in-person versus virtual attendance. DESIGN: Cross-sectional survey-based evaluation. SETTING: International comprehensive cleft care workshop. PARTICIPANTS: Total of 489 participants. INTERVENTIONS: Three-day simulation-based hybrid comprehensive cleft care workshop. MAIN OUTCOME MEASURES: Participant demographic data, perceived barriers and interventions needed for global comprehensive cleft care delivery, participant workshop satisfaction, and perceived short-term impact on practice stratified by in-person versus virtual attendance. RESULTS: The workshop included 489 participants from 5 continents. The response rate was 39.9%. Participants perceived financial factors (30.3%) the most significant barrier and improvement in training (39.8%) as the most important intervention to overcome barriers facing cleft care delivery in low to middle-income countries. All participants reported a high level of satisfaction with the workshop and a strong positive perceived short-term impact on their practice. Importantly, while this was true for both in-person and virtual attendees, in-person attendees reported a significantly higher satisfaction with the workshop (28.63 ± 3.08 vs 27.63 ± 3.93; P = .04) and perceived impact on their clinical practice (22.37 ± 3.42 vs 21.02 ± 3.45 P = .01). CONCLUSION: Hybrid simulation-based educational comprehensive cleft care workshops are overall well received by participants and have a positive perceived impact on their clinical practices. In-person attendance is associated with significantly higher satisfaction and perceived impact on practice. Considering that financial and health constraints may limit live meeting attendance, future efforts will focus on making in-person and virtual attendance more comparable.

Targeting HIV-1 Reverse Transcriptase Using a Fragment-Based Approach.

Human immunodeficiency virus type I (HIV-1) is a retrovirus that infects cells of the host's immune system leading to acquired immunodeficiency syndrome and potentially death. Although treatments are available to prevent its progression, HIV-1 remains a major burden on health resources worldwide. Continued emergence of drug-resistance mutations drives the need for novel drugs that can inhibit HIV-1 replication through new pathways. The viral protein reverse transcriptase (RT) plays a fundamental role in the HIV-1 replication cycle, and multiple approved medications target this enzyme. In this study, fragment-based drug discovery was used to optimize a previously identified hit fragment (compound B-1), which bound RT at a novel site. Three series of compounds were synthesized and evaluated for their HIV-1 RT binding and inhibition. These series were designed to investigate different vectors around the initial hit in an attempt to improve inhibitory activity against RT. Our results show that the 4-position of the core scaffold is important for binding of the fragment to RT, and a lead compound with a cyclopropyl substitution was selected and further investigated. Requirements for binding to the NNRTI-binding pocket (NNIBP) and a novel adjacent site were investigated, with lead compound 27-a minimal but efficient NNRTI-offering a starting site for the development of novel dual NNIBP-Adjacent site inhibitors.

Conscientious Objection and Institutional Objection to Voluntary Assistance in Dying: An Ethico-legal Critique.

This column examines conscientious objection and institutional objection in Australian voluntary assistance in dying. It reviews the current legislative regimes and then examines these practices from an ethical perspective, and raises particular concerns and suggestions with how conscientious objection and institutional objection should be operationalised.

The Caudal ParaHox gene is required for hindgut development in the mollusc Tritia (a.k.a. Ilyanassa).

Caudal homeobox genes are found across animals, typically linked to two other homeobox genes in what has been called the ParaHox cluster. These genes have been proposed to pattern the anterior-posterior axis of the endoderm ancestrally, but the expression of Caudal in extant groups is varied and often occurs in other germ layers. Here we examine the role of Caudal in the embryo of the mollusc Tritia (Ilyanassa) obsoleta. ToCaudal expression is initially broad, then becomes progressively restricted and is finally only in the developing hindgut (a.k.a. intestine). Knockdown of ToCaudal using morpholino oligonucleotides specifically blocks hindgut development, indicating that despite its initially broad expression, the functional role of ToCaudal is in hindgut patterning. This is the first functional characterization of Caudal in an animal with spiralian development, which is an ancient mode of embryogenesis that arose early in bilaterian animal evolution. These results are consistent with the hypothesis that the ancestral role of the ParaHox genes was anterior-posterior patterning of the endoderm.