Gastric adenocarcinoma location and postoperative complication rates in Asian patients: A 2014-2019 NSQIP analysis.

BACKGROUND: Asian gastric cancer patients have higher long-term survival rates post-gastrectomy. This study compares 30-day post-gastrectomy outcomes between Asians and non-Asians. METHODS: Gastric cancer patients undergoing elective gastrectomies were identified in 2014-2019 NSQIP datasets (n â€‹= â€‹1,438). Demographics, comorbidities, and postoperative outcomes were analyzed. RESULTS: Asians had lower odds of total gastrectomy (AOR â€‹= â€‹0.52, p â€‹= â€‹0.003), age ≥65 (AOR â€‹= â€‹0.60, p â€‹= â€‹0.006), smoking history (AOR â€‹= â€‹0.35, p â€‹< â€‹0.001), dyspnea (AOR â€‹= â€‹0.25, p â€‹= â€‹0.01), and hypoalbuminemia (AOR â€‹= â€‹0.62, p â€‹= â€‹0.025); they also had lower BMI (p â€‹< â€‹0.001). Postoperative outcomes were not significantly different aside from a shorter median length of hospital stay in days (LOS) (Asians: 7 (6, 11); non-Asians: 8 (6, 11); p â€‹< â€‹0.001). CONCLUSIONS: Asian gastric cancer patients have significantly lower odds of having select preoperative comorbidities and have shorter hospital LOS.

Targeting the non-coding genome and temozolomide signature enables CRISPR-mediated glioma oncolysis.

Glioblastoma (GBM) is the most common lethal primary brain cancer in adults. Despite treatment regimens including surgical resection, radiotherapy, and temozolomide (TMZ) chemotherapy, growth of residual tumor leads to therapy resistance and death. At recurrence, a quarter to a third of all gliomas have hypermutated genomes, with mutational burdens orders of magnitude greater than in normal tissue. Here, we quantified the mutational landscape progression in a patient's primary and recurrent GBM, and we uncovered Cas9-targetable repeat elements. We show that CRISPR-mediated targeting of highly repetitive loci enables rapid elimination of GBM cells, an approach we term "genome shredding." Importantly, in the patient's recurrent GBM, we identified unique repeat sequences with TMZ mutational signature and demonstrated that their CRISPR targeting enables cancer-specific cell ablation. "Cancer shredding" leverages the non-coding genome and therapy-induced mutational signatures for targeted GBM cell depletion and provides an innovative paradigm to develop treatments for hypermutated glioma.

Endogenous spacing enables co-processing of microRNAs and efficient combinatorial RNAi.

We present Multi-miR, a microRNA-embedded shRNA system modeled after endogenous microRNA clusters that enables simultaneous expression of up to three or four short hairpin RNAs (shRNAs) from a single promoter without loss of activity, enabling robust combinatorial RNA interference (RNAi). We further developed complementary all-in-one vectors that are over one log-scale more sensitive to doxycycline-mediated activation in vitro than previous methods and resistant to shRNA inactivation in vivo. We demonstrate the utility of this system for intracranial expression of shRNAs in a glioblastoma model. Additionally, we leverage this platform to target the redundant RAF signaling node in a mouse model of KRAS-mutant cancer and show that robust combinatorial synthetic lethality efficiently abolishes tumor growth.

Enhanced BRAF engagement by NRAS mutants capable of promoting melanoma initiation.

A distinct profile of NRAS mutants is observed in each tumor type. It is unclear whether these profiles are determined by mutagenic events or functional differences between NRAS oncoproteins. Here, we establish functional hallmarks of NRAS mutants enriched in human melanoma. We generate eight conditional, knock-in mouse models and show that rare melanoma mutants (NRAS G12D, G13D, G13R, Q61H, and Q61P) are poor drivers of spontaneous melanoma formation, whereas common melanoma mutants (NRAS Q61R, Q61K, or Q61L) induce rapid tumor onset with high penetrance. Molecular dynamics simulations, combined with cell-based protein-protein interaction studies, reveal that melanomagenic NRAS mutants form intramolecular contacts that enhance BRAF binding affinity, BRAF-CRAF heterodimer formation, and MAPK > ERK signaling. Along with the allelic series of conditional mouse models we describe, these results establish a mechanistic basis for the enrichment of specific NRAS mutants in human melanoma.

CRISPR-Cas9-mediated nuclear transport and genomic integration of nanostructured genes in human primary cells.

DNA nanostructures are a promising tool to deliver molecular payloads to cells. DNA origami structures, where long single-stranded DNA is folded into a compact nanostructure, present an attractive approach to package genes; however, effective delivery of genetic material into cell nuclei has remained a critical challenge. Here, we describe the use of DNA nanostructures encoding an intact human gene and a fluorescent protein encoding gene as compact templates for gene integration by CRISPR-mediated homology-directed repair (HDR). Our design includes CRISPR-Cas9 ribonucleoprotein binding sites on DNA nanostructures to increase shuttling into the nucleus. We demonstrate efficient shuttling and genomic integration of DNA nanostructures using transfection and electroporation. These nanostructured templates display lower toxicity and higher insertion efficiency compared to unstructured double-stranded DNA templates in human primary cells. Furthermore, our study validates virus-like particles as an efficient method of DNA nanostructure delivery, opening the possibility of delivering nanostructures in vivo to specific cell types. Together, these results provide new approaches to gene delivery with DNA nanostructures and establish their use as HDR templates, exploiting both their design features and their ability to encode genetic information. This work also opens a door to translate other DNA nanodevice functions, such as biosensing, into cell nuclei.

Comprehensive deletion landscape of CRISPR-Cas9 identifies minimal RNA-guided DNA-binding modules.

Proteins evolve through the modular rearrangement of elements known as domains. Extant, multidomain proteins are hypothesized to be the result of domain accretion, but there has been limited experimental validation of this idea. Here, we introduce a technique for genetic minimization by iterative size-exclusion and recombination (MISER) for comprehensively making all possible deletions of a protein. Using MISER, we generate a deletion landscape for the CRISPR protein Cas9. We find that the catalytically-dead Streptococcus pyogenes Cas9 can tolerate large single deletions in the REC2, REC3, HNH, and RuvC domains, while still functioning in vitro and in vivo, and that these deletions can be stacked together to engineer minimal, DNA-binding effector proteins. In total, our results demonstrate that extant proteins retain significant modularity from the accretion process and, as genetic size is a major limitation for viral delivery systems, establish a general technique to improve genome editing and gene therapy-based therapeutics.

Variables Associated with Coronavirus Disease 2019 Vaccine Hesitancy Amongst Patients with Neurological Disorders.

INTRODUCTION: Given that the success of vaccines against coronavirus disease 2019 (COVID-19) relies on herd immunity, identifying patients at risk for vaccine hesitancy is imperative-particularly for those at high risk for severe COVID-19 (i.e., minorities and patients with neurological disorders). METHODS: Among patients from a large neuroscience institute in Hawaii, vaccine hesitancy was investigated in relation to over 30 sociodemographic variables and medical comorbidities, via a telephone quality improvement survey conducted between 23 January 2021 and 13 February 2021. RESULTS: Vaccine willingness (n = 363) was 81.3%. Univariate analysis identified that the odds of vaccine acceptance reduced for patients who do not regard COVID-19 as a severe illness, are of younger age, have a lower Charlson Comorbidity Index, use illicit drugs, or carry Medicaid insurance. Multivariable logistic regression identified the best predictors of vaccine hesitancy to be: social media use to obtain COVID-19 information, concerns regarding vaccine safety, self-perception of a preexisting medical condition contraindicated with vaccination, not having received the annual influenza vaccine, having some high school education only, being a current smoker, and not having a prior cerebrovascular accident. Unique amongst males, a conservative political view strongly predicted vaccine hesitancy. Specifically for Asians, a higher body mass index, while for Native Hawaiians and other Pacific Islanders (NHPI), a positive depression screen, both reduced the odds of vaccine acceptance. CONCLUSION: Upon identifying the variables associated with vaccine hesitancy amongst patients with neurological disorders, our clinic is now able to efficiently provide ancillary COVID-19 education to sub-populations at risk for vaccine hesitancy. While our results may be limited to the sub-population of patients with neurological disorders, the findings nonetheless provide valuable insight to understanding vaccine hesitancy.

PI5P4Ks drive metabolic homeostasis through peroxisome-mitochondria interplay.

PI5P4Ks are a class of phosphoinositide kinases that phosphorylate PI-5-P to PI-4,5-P2. Distinct localization of phosphoinositides is fundamental for a multitude of cellular functions. Here, we identify a role for peroxisomal PI-4,5-P2 generated by the PI5P4Ks in maintaining energy balance. We demonstrate that PI-4,5-P2 regulates peroxisomal fatty acid oxidation by mediating trafficking of lipid droplets to peroxisomes, which is essential for sustaining mitochondrial metabolism. Using fluorescent-tagged lipids and metabolite tracing, we show that loss of the PI5P4Ks significantly impairs lipid uptake and ß-oxidation in the mitochondria. Further, loss of PI5P4Ks results in dramatic alterations in mitochondrial structural and functional integrity, which under nutrient deprivation is further exacerbated, causing cell death. Notably, inhibition of the PI5P4Ks in cancer cells and mouse tumor models leads to decreased cell viability and tumor growth, respectively. Together, these studies reveal an unexplored role for PI5P4Ks in preserving metabolic homeostasis, which is necessary for tumorigenesis.

Cancer-specific loss of TERT activation sensitizes glioblastoma to DNA damage.

Most glioblastomas (GBMs) achieve cellular immortality by acquiring a mutation in the telomerase reverse transcriptase (TERT) promoter. TERT promoter mutations create a binding site for a GA binding protein (GABP) transcription factor complex, whose assembly at the promoter is associated with TERT reactivation and telomere maintenance. Here, we demonstrate increased binding of a specific GABPB1L-isoform-containing complex to the mutant TERT promoter. Furthermore, we find that TERT promoter mutant GBM cells, unlike wild-type cells, exhibit a critical near-term dependence on GABPB1L for proliferation, notably also posttumor establishment in vivo. Up-regulation of the protein paralogue GABPB2, which is normally expressed at very low levels, can rescue this dependence. More importantly, when combined with frontline temozolomide (TMZ) chemotherapy, inducible GABPB1L knockdown and the associated TERT reduction led to an impaired DNA damage response that resulted in profoundly reduced growth of intracranial GBM tumors. Together, these findings provide insights into the mechanism of cancer-specific TERT regulation, uncover rapid effects of GABPB1L-mediated TERT suppression in GBM maintenance, and establish GABPB1L inhibition in combination with chemotherapy as a therapeutic strategy for TERT promoter mutant GBM.

Site-Specific Bioconjugation through Enzyme-Catalyzed Tyrosine-Cysteine Bond Formation.

The synthesis of protein-protein and protein-peptide conjugates is an important capability for producing vaccines, immunotherapeutics, and targeted delivery agents. Herein we show that the enzyme tyrosinase is capable of oxidizing exposed tyrosine residues into o-quinones that react rapidly with cysteine residues on target proteins. This coupling reaction occurs under mild aerobic conditions and has the rare ability to join full-size proteins in under 2 h. The utility of the approach is demonstrated for the attachment of cationic peptides to enhance the cellular delivery of CRISPR-Cas9 20-fold and for the coupling of reporter proteins to a cancer-targeting antibody fragment without loss of its cell-specific binding ability. The broad applicability of this technique provides a new building block approach for the synthesis of protein chimeras.

ATF4 Regulates MYB to Increase γ-Globin in Response to Loss of ß-Globin.

ß-Hemoglobinopathies can trigger rapid production of red blood cells in a process known as stress erythropoiesis. Cellular stress prompts differentiating erythroid precursors to express high levels of fetal γ-globin. However, the mechanisms underlying γ-globin production during cellular stress are still poorly defined. Here, we use CRISPR-Cas genome editing to model the stress caused by reduced levels of adult ß-globin. We find that decreased ß-globin is sufficient to induce robust re-expression of γ-globin, and RNA sequencing (RNA-seq) of differentiating isogenic erythroid precursors implicates ATF4 as a causal regulator of this response. ATF4 binds within the HBS1L-MYB intergenic enhancer and regulates expression of MYB, a known γ-globin regulator. Overall, the reduction of ATF4 upon ß-globin knockout decreases the levels of MYB and BCL11A. Identification of ATF4 as a key regulator of globin compensation adds mechanistic insight to the poorly understood phenomenon of stress-induced globin compensation and could inform strategies to treat hemoglobinopathies.

Potent CRISPR-Cas9 inhibitors from Staphylococcus genomes.

Anti-CRISPRs (Acrs) are small proteins that inhibit the RNA-guided DNA targeting activity of CRISPR-Cas enzymes. Encoded by bacteriophage and phage-derived bacterial genes, Acrs prevent CRISPR-mediated inhibition of phage infection and can also block CRISPR-Cas-mediated genome editing in eukaryotic cells. To identify Acrs capable of inhibiting Staphylococcus aureus Cas9 (SauCas9), an alternative to the most commonly used genome editing protein Streptococcus pyogenes Cas9 (SpyCas9), we used both self-targeting CRISPR screening and guilt-by-association genomic search strategies. Here we describe three potent inhibitors of SauCas9 that we name AcrIIA13, AcrIIA14, and AcrIIA15. These inhibitors share a conserved N-terminal sequence that is dispensable for DNA cleavage inhibition and have divergent C termini that are required in each case for inhibition of SauCas9-catalyzed DNA cleavage. In human cells, we observe robust inhibition of SauCas9-induced genome editing by AcrIIA13 and moderate inhibition by AcrIIA14 and AcrIIA15. We also find that the conserved N-terminal domain of AcrIIA13-AcrIIA15 binds to an inverted repeat sequence in the promoter of these Acr genes, consistent with its predicted helix-turn-helix DNA binding structure. These data demonstrate an effective strategy for Acr discovery and establish AcrIIA13-AcrIIA15 as unique bifunctional inhibitors of SauCas9.

Structural basis for AcrVA4 inhibition of specific CRISPR-Cas12a.

CRISPR-Cas systems provide bacteria and archaea with programmable immunity against mobile genetic elements. Evolutionary pressure by CRISPR-Cas has driven bacteriophage to evolve small protein inhibitors, anti-CRISPRs (Acrs), that block Cas enzyme function by wide-ranging mechanisms. We show here that the inhibitor AcrVA4 uses a previously undescribed strategy to recognize the L. bacterium Cas12a (LbCas12a) pre-crRNA processing nuclease, forming a Cas12a dimer, and allosterically inhibiting DNA binding. The Ac. species Cas12a (AsCas12a) enzyme, widely used for genome editing applications, contains an ancestral helical bundle that blocks AcrVA4 binding and allows it to escape anti-CRISPR recognition. Using biochemical, microbiological, and human cell editing experiments, we show that Cas12a orthologs can be rendered either sensitive or resistant to AcrVA4 through rational structural engineering informed by evolution. Together, these findings explain a new mode of CRISPR-Cas inhibition and illustrate how structural variability in Cas effectors can drive opportunistic co-evolution of inhibitors by bacteriophage.

Dengue outbreaks in Hawai'i After WWII - A Review of Public Health Response and Scientific Literature.

The four serotypes of dengue virus (DENV) cause the most important and common arthropod-borne viral diseases in humans. There have been three major dengue outbreaks in Hawai'i since 1946. The most recent and largest outbreak occurred on Hawai'i Island in 2015-2016. This article reviews the public health response to dengue outbreaks over the period 2001-2016, as well as scientific literature on dengue outbreaks in Hawai'i. As summarized in the assessment by the Centers for Disease Control and Prevention in 2015, Hawaii's response to the dengue outbreak was timely, appropriate, and well-coordinated. All facets of a public health response to the outbreak were adequately addressed, but communications and medical entomologic capacities could be improved. The observations of Aedes aegypti on Hawai'i Island and of its co-localization with confirmed human cases highlight the importance of continuous vector surveillance and entomologic research. In-depth studies on the molecular epidemiology, entomology, and epidemiological investigation would provide new insights into the latest outbreak and into strategies to combat DENV and other arboviruses in the future.

Postoperative sternal dehiscence in obese patients: incidence and prevention.

BACKGROUND: Obesity has been identified as the single most important risk factor for postoperative sternal infection in coronary bypass surgery patients. It is also a major risk factor for sternal dehiscence, with or without infection, for any type of cardiac operation. We assessed whether prophylactic measures could prevent this complication. METHODS: Two studies were conducted. In study A, 3,158 heart surgery patients were analyzed at 3 cardiac units. Obesity was defined as body mass index (BMI) more than 30. Group I (1,253 obese [39.7%]) was compared with group II (1,905 nonobese [60.3%]). Sternal closure was done at the surgeon's preference: (a) plain wires through and through the bone; (b) peristernal figure-of-eight wires; or (c) peristernal method, using stainless-steel cables. In study B, 123 obese patients were prospectively divided into 2 subgroups. Group B-1 (54 patients) underwent lateral prophylactic sternal reinforcement before placement of peristernal wires. Group B-2 (69 patients) had standard sternal closure, as in study A. RESULTS: In study A, group I had 81 dehiscences (6.46%); 78 also suffered deep sternal infection and mediastinitis (96%). Despite treatment, dehiscence recurred in 13, and mortality was 38.4%. In group II nonobese patients, 31 dehisced (1.6%, p = 0.000), with no mortality. In study B, group B-1 (54) had 0% dehiscence versus group B-2 (69) with 6 dehiscences (8.7%). CONCLUSIONS: In our study, the rate of obesity is high ( approximately 40%). Sternal dehiscence is real when the BMI is more than 30 (6.46%), and has high morbidity and mortality. Prophylactic sternal reinforcement seems to prevent this complication.