Neutrophil-mediated innate immune resistance to bacterial pneumonia is dependent on Tet2 function.

Individuals with clonal hematopoiesis of indeterminate potential (CHIP) are at increased risk of aging related health conditions and all-cause mortality, but whether CHIP impacts risk of infection is much less clear. Using UK Biobank data, we revealed a positive association between CHIP and incident pneumonia in 438,421 individuals. We show that inflammation enhanced pneumonia risk, as CHIP carriers with a hypomorphic IL6 receptor polymorphism were protected. To better characterize the pathways of susceptibility, we challenged hematopoietic Tet Methylcytosine Dioxygenase 2 knockout (Tet2-/-) and floxed control mice (Tet2f/f) with Streptococcus pneumoniae. As with human CHIP carriers, Tet2-/- mice had hematopoietic abnormalities resulting in the expansion of inflammatory monocytes and neutrophils in peripheral blood. Yet, these cells were insufficient in defending against S. pneumoniae and resulted in increased pathology, impaired bacterial clearance, and higher mortality in Tet2-/- mice. We delineated the transcriptional landscape of Tet2-/- neutrophils and found that while inflammation-related pathways were upregulated in Tet2-/- neutrophils, migration and motility pathways were compromised. Using live-imaging techniques, we demonstrated impairments in motility, pathogen uptake and neutrophil extracellular trap (NET) formation by Tet2-/- neutrophils. Collectively, we show that CHIP is a risk factor for bacterial pneumonia related to innate immune impairments.

Comparing a Portable Motion Analysis System against the Gold Standard for Potential Anterior Cruciate Ligament Injury Prevention and Screening.

Knee kinematics during a drop vertical jump, measured by the Kinect V2 (Microsoft, Redmond, WA, USA), have been shown to be associated with an increased risk of non-contact anterior cruciate ligament injury. The accuracy and reliability of the Microsoft Kinect V2 has yet to be assessed specifically for tracking the coronal and sagittal knee angles of the drop vertical jump. Eleven participants performed three drop vertical jumps that were recorded using both the Kinect V2 and a gold standard motion analysis system (Vicon, Los Angeles, CA, USA). The initial coronal, peak coronal, and peak sagittal angles of the left and right knees were measured by both systems simultaneously. Analysis of the data obtained by the Kinect V2 was performed by our software. The differences in the mean knee angles measured by the Kinect V2 and the Vicon system were non-significant for all parameters except for the peak sagittal angle of the right leg with a difference of 7.74 degrees and a p-value of 0.008. There was excellent agreement between the Kinect V2 and the Vicon system, with intraclass correlation coefficients consistently over 0.75 for all knee angles measured. Visual analysis revealed a moderate frame-to-frame variability for coronal angles measured by the Kinect V2. The Kinect V2 can be used to capture knee coronal and sagittal angles with sufficient accuracy during a drop vertical jump, suggesting that a Kinect-based portable motion analysis system is suitable to screen individuals for the risk of non-contact anterior cruciate ligament injury.

Sex differences in sleep architecture in a mouse model of Huntington's disease.

Sleep and circadian rhythm disturbances are common features of Huntington's disease (HD). HD is an autosomal dominant neurodegenerative disorder that affects men and women in equal numbers, but some epidemiological studies as well as preclinical work indicate there may be sex differences in disease presentation and progression. Since sex differences in HD could provide important insights to understand cellular and molecular mechanism(s), we used the bacterial artificial chromosome transgenic mouse model of HD (BACHD) to examine whether sex differences in sleep/wake cycles are detectable in an animal model of the disease. Electroencephalography/electromyography (EEG/EMG) was used to measure sleep/wake states and polysomnographic patterns in young adult (12-week-old) male and female wild-type and BACHD mice. Our findings show that male, but not female, BACHD mice exhibited increased variation in phases of the rhythms as compared to age- and sex-matched wild-types. For both rapid-eye movement (REM) and non-rapid eye movement (NREM) sleep, genotypic and sex differences were detected. In particular, the BACHD males spent less time in NREM sleep and exhibited a more fragmented sleep than the other groups. Finally, in response to 6 h of sleep deprivation, both genotypes and sexes displayed the predicted homeostatic responses to sleep loss. These findings suggest that females are relatively protected early in disease progression in this HD model.

Prime editing using CRISPR-Cas12a and circular RNAs in human cells.

Genome editing with prime editors based on CRISPR-Cas9 is limited by the large size of the system and the requirement for a G/C-rich protospacer-adjacent motif (PAM) sequence. Here, we use the smaller Cas12a protein to develop four circular RNA-mediated prime editor (CPE) systems: nickase-dependent CPE (niCPE), nuclease-dependent CPE (nuCPE), split nickase-dependent CPE (sniCPE) and split nuclease-dependent CPE (snuCPE). CPE systems preferentially recognize T-rich genomic regions and possess a potential multiplexing capacity in comparison to corresponding Cas9-based systems. The efficiencies of the nuclease-based systems are up to 10.42%, whereas niCPE and sniCPE reach editing frequencies of up to 24.89% and 40.75% without positive selection in human cells, respectively. A derivative system, called one-sniCPE, combines all three RNA editing components under a single promoter. By arraying CRISPR RNAs for different targets in one circular RNA, we also demonstrate low-efficiency editing of up to four genes simultaneously with the nickase prime editors niCPE and sniCPE.

Outcomes of ventriculoperitoneal shunt placement with or without laparoscopic assistance: An analysis of the national inpatient sample.

Background: Ventriculoperitoneal shunt (VPS) can be placed solely by a neurosurgeon often via an open-laparotomy approach, or laparoscopically as a collaborative effort between a neurosurgeon and a general surgeon. Prior studies have shown conflicting results when examining outcomes regarding infection, revision rate, hospital charges, length of stay, and mortality between the open mini-laparotomy and the laparoscopic approaches. Objective: The current study uses the National Inpatient Sample (NIS) to compare outcomes of open mini-laparotomy vs. laparoscopic collaborative approach in VPS placement. Methods: We performed a retrospective database study of the NIS from October 2015-December 2017 utilizing International Classification of Diseases, 10th Revision coding to identify all cases of VPS placement. All analyses accounted for the sampling design of the NIS. Results: A total of 6580 cases (4969 with open mini-laparotomy approach and 1611 with laparoscopic collaborative approach) met inclusion criteria. Hospital charges, infection rates, and revision rates were similar between approaches. There were no significant differences in length of stay, mortality, or complication rates between the two approaches. Conclusion: The collaborative, laparoscopic approach to VPS placement has similar outcomes and is non-inferior to the traditional open mini-laparotomy approach.

Precise integration of large DNA sequences in plant genomes using PrimeRoot editors.

A technique for chromosomal insertion of large DNA segments is much needed in plant breeding and synthetic biology to facilitate the introduction of desired agronomic traits and signaling and metabolic pathways. Here we describe PrimeRoot, a genome editing approach to generate targeted precise large DNA insertions in plants. Third-generation PrimeRoot editors employ optimized prime editing guide RNA designs, an enhanced plant prime editor and superior recombinases to enable precise large DNA insertions of up to 11.1 kilobases into plant genomes. We demonstrate the use of PrimeRoot to accurately introduce gene regulatory elements in rice. In this study, we also integrated a gene cassette comprising PigmR, which confers rice blast resistance driven by an Act1 promoter, into a predicted genomic safe harbor site of Kitaake rice and obtain edited plants harboring the expected insertion with an efficiency of 6.3%. We found that these rice plants have increased blast resistance. These results establish PrimeRoot as a promising approach to precisely insert large segments of DNA in plants.

Managing Cardiovascular Risk in Systemic Lupus Erythematosus: Considerations for the Clinician.

A significant improvement in the survival of patients with systemic lupus erythematosus (SLE) over recent decades is largely attributed to the impact of disease-modifying therapies on end-organ damage. Thus, cardiovascular disease now represents the leading cause of mortality in SLE. Various disease-specific mechanisms are responsible for advanced atherosclerosis, as they lead to premature endothelial dysfunction, arterial stiffness, arterial wall thickening, and plaque formation. Consequently, in the assessment of cardiovascular risk in SLE, we must not only consider traditional risk factors (ie, age, gender, dyslipidemia) but also the additional role of non-traditional risk factors such as persistent disease activity and prolonged corticosteroid use. Cardiovascular risk assessment incorporates general cardiovascular screening, as existing risk prediction scores underestimate cardiovascular risk in this patient population. There is also an expanding role of imaging modalities in screening. Risk reduction strategies integrate unique considerations for the use of low-dose aspirin and more stringent hypertension targets. Hydroxychloroquine is the only disease-modifying therapy with known cardiovascular benefit in SLE, though this is a promising area of study.

Assigning functionality to cysteines by base editing of cancer dependency genes.

Covalent chemistry represents an attractive strategy for expanding the ligandability of the proteome, and chemical proteomics has revealed numerous electrophile-reactive cysteines on diverse human proteins. Determining which of these covalent binding events affect protein function, however, remains challenging. Here we describe a base-editing strategy to infer the functionality of cysteines by quantifying the impact of their missense mutation on cancer cell proliferation. The resulting atlas, which covers more than 13,800 cysteines on more than 1,750 cancer dependency proteins, confirms the essentiality of cysteines targeted by covalent drugs and, when integrated with chemical proteomic data, identifies essential, ligandable cysteines in more than 160 cancer dependency proteins. We further show that a stereoselective and site-specific ligand targeting an essential cysteine in TOE1 inhibits the nuclease activity of this protein through an apparent allosteric mechanism. Our findings thus describe a versatile method and valuable resource to prioritize the pursuit of small-molecule probes with high function-perturbing potential.

Strand-preferred base editing of organellar and nuclear genomes using CyDENT.

Transcription-activator-like effector (TALE)-based tools for base editing of nuclear and organellar DNA rely on double-stranded DNA deaminases, which edit substrate bases on both strands of DNA, reducing editing precision. Here, we present CyDENT base editing, a CRISPR-free, strand-selective, modular base editor. CyDENT comprises a pair of TALEs fused with a FokI nickase, a single-strand-specific cytidine deaminase and an exonuclease to generate a single-stranded DNA substrate for deamination. We demonstrate effective base editing in nuclear, mitochondrial and chloroplast genomes. At certain mitochondrial sites, we show editing efficiencies of 14% and strand specificity of 95%. Furthermore, by exchanging the CyDENT deaminase with one that prefers editing GC motifs, we demonstrate up to 20% mitochondrial base editing at sites that are otherwise inaccessible to editing by other methods. The modular nature of CyDENT enables a suite of bespoke base editors for various applications.

Discovery of deaminase functions by structure-based protein clustering.

The elucidation of protein function and its exploitation in bioengineering have greatly advanced the life sciences. Protein mining efforts generally rely on amino acid sequences rather than protein structures. We describe here the use of AlphaFold2 to predict and subsequently cluster an entire protein family based on predicted structure similarities. We selected deaminase proteins to analyze and identified many previously unknown properties. We were surprised to find that most proteins in the DddA-like clade were not double-stranded DNA deaminases. We engineered the smallest single-strand-specific cytidine deaminase, enabling efficient cytosine base editor (CBE) to be packaged into a single adeno-associated virus (AAV). Importantly, we profiled a deaminase from this clade that edits robustly in soybean plants, which previously was inaccessible to CBEs. These discovered deaminases, based on AI-assisted structural predictions, greatly expand the utility of base editors for therapeutic and agricultural applications.

Sex Differences in Sleep Phenotypes in the BACHD Mouse Model of Huntington's Disease.

Sleep and circadian rhythm disturbances are common features of Huntington's disease (HD). HD is an autosomal dominant neurodegenerative disorder that affects men and women in equal numbers, but some epidemiological studies as well as preclinical work indicate there may be sex differences in disease progression. Since sex differences in HD could provide important insights to understand cellular and molecular mechanism(s), we used the bacterial artificial chromosome transgenic mouse model of HD (BACHD) to examine whether sex differences in sleep/wake cycles are detectable in an animal model of the disease. Electroencephalography/electromyography (EEG/EMG) was used to measure sleep/wake states and polysomnographic patterns in young adult (12 week-old) male and female wild-type and BACHD mice. Our findings show that male, but not female, BACHD mice exhibited increased variation in phases of the rhythms as compared to age and sex matched wild-types. For both Rapid-eye movement (REM) and Non-rapid eye movement (NREM) sleep, genotypic and sex differences were detected. In particular, the BACHD males spent less time in NREM and exhibited a more fragmented sleep than the other groups. Both male and female BACHD mice exhibited significant changes in delta but not in gamma power compared to wild-type mice. Finally, in response to a 6-hrs sleep deprivation, both genotypes and sexes displayed predicted homeostatic responses to sleep loss. These findings suggest that females are relatively protected early in disease progression in this HD model.

Tuning plant phenotypes by precise, graded downregulation of gene expression.

The ability to control gene expression and generate quantitative phenotypic changes is essential for breeding new and desired traits into crops. Here we report an efficient, facile method for downregulating gene expression to predictable, desired levels by engineering upstream open reading frames (uORFs). We used base editing or prime editing to generate de novo uORFs or to extend existing uORFs by mutating their stop codons. By combining these approaches, we generated a suite of uORFs that incrementally downregulate the translation of primary open reading frames (pORFs) to 2.5-84.9% of the wild-type level. By editing the 5' untranslated region of OsDLT, which encodes a member of the GRAS family and is involved in the brassinosteroid transduction pathway, we obtained, as predicted, a series of rice plants with varied plant heights and tiller numbers. These methods offer an efficient way to obtain genome-edited plants with graded expression of traits.

Acute organ injury and long-term sequelae of severe pneumococcal infections.

Streptococcus pneumoniae (Spn) is a major public health problem, as it is a main cause of otitis media, community-acquired pneumonia, bacteremia, sepsis, and meningitis. Acute episodes of pneumococcal disease have been demonstrated to cause organ damage with lingering negative consequences. Cytotoxic products released by the bacterium, biomechanical and physiological stress resulting from infection, and the corresponding inflammatory response together contribute to organ damage accrued during infection. The collective result of this damage can be acutely life-threatening, but among survivors, it also contributes to the long-lasting sequelae of pneumococcal disease. These include the development of new morbidities or exacerbation of pre-existing conditions such as COPD, heart disease, and neurological impairments. Currently, pneumonia is ranked as the 9th leading cause of death, but this estimate only considers short-term mortality and likely underestimates the true long-term impact of disease. Herein, we review the data that indicates damage incurred during acute pneumococcal infection can result in long-term sequelae which reduces quality of life and life expectancy among pneumococcal disease survivors.

Base editing rescue of spinal muscular atrophy in cells and in mice.

Spinal muscular atrophy (SMA), the leading genetic cause of infant mortality, arises from survival motor neuron (SMN) protein insufficiency resulting from SMN1 loss. Approved therapies circumvent endogenous SMN regulation and require repeated dosing or may wane. We describe genome editing of SMN2, an insufficient copy of SMN1 harboring a C6>T mutation, to permanently restore SMN protein levels and rescue SMA phenotypes. We used nucleases or base editors to modify five SMN2 regulatory regions. Base editing converted SMN2 T6>C, restoring SMN protein levels to wild type. Adeno-associated virus serotype 9-mediated base editor delivery in Δ7SMA mice yielded 87% average T6>C conversion, improved motor function, and extended average life span, which was enhanced by one-time base editor and nusinersen coadministration (111 versus 17 days untreated). These findings demonstrate the potential of a one-time base editing treatment for SMA.

A molecular glue approach to control the half-life of CRISPR-based technologies.

Cas9 is a programmable nuclease that has furnished transformative technologies, including base editors and transcription modulators (e.g., CRISPRi/a), but several applications of these technologies, including therapeutics, mandatorily require precision control of their half-life. For example, such control can help avert any potential immunological and adverse events in clinical trials. Current genome editing technologies to control the half-life of Cas9 are slow, have lower activity, involve fusion of large response elements (> 230 amino acids), utilize expensive controllers with poor pharmacological attributes, and cannot be implemented in vivo on several CRISPR-based technologies. We report a general platform for half-life control using the molecular glue, pomalidomide, that binds to a ubiquitin ligase complex and a response-element bearing CRISPR-based technology, thereby causing the latter's rapid ubiquitination and degradation. Using pomalidomide, we were able to control the half-life of large CRISPR-based technologies (e.g., base editors, CRISPRi) and small anti-CRISPRs that inhibit such technologies, allowing us to build the first examples of on-switch for base editors. The ability to switch on, fine-tune and switch-off CRISPR-based technologies with pomalidomide allowed complete control over their activity, specificity, and genome editing outcome. Importantly, the miniature size of the response element and favorable pharmacological attributes of the drug pomalidomide allowed control of activity of base editor in vivo using AAV as the delivery vehicle. These studies provide methods and reagents to precisely control the dosage and half-life of CRISPR-based technologies, propelling their therapeutic development.

Prioritization of head and neck cancer patient care during the COVID-19 pandemic: a retrospective cohort study.

BACKGROUND: The COVID-19 pandemic placed considerable strain on the healthcare system, leading to the re-allocation of resources and implementation of new practice guidelines. The objective of this study is to assess the impact of COVID-19 guideline modifications on head and neck cancer (HNC) care at two tertiary care centers in Canada. METHODS: A retrospective cohort study was conducted. HNC patients seen at two tertiary care centers before and after the onset of the COVID-19 pandemic (pre-pandemic: July 1st, 2019, to February 29th, 2020; pandemic: March 1st, 2020, to October 31st, 2020) were included. The pre-pandemic and pandemic cohorts were compared according to patient and tumor characteristics, duration of HNC workup, and treatment type and duration. Mean differences in cancer care wait times, including time to diagnosis, tumor board, and treatment as well as total treatment package time and postoperative hospital stay were compared between cohorts. Univariate and multivariate analyses were used to compare characteristics and outcomes between cohorts. RESULTS: Pre-pandemic (n = 132) and pandemic (n = 133) patients did not differ significantly in sex, age, habits, or tumor characteristics. The percentage of patients who received surgery only, chemo/radiotherapy (CXRT) only, and surgery plus adjuvant CXRT did not differ significantly between cohorts. Pandemic patients experienced a significant time reduction compared to pre-pandemic patients with regards to the date first seen by a HNC service until start of treatment ([Formula: see text] = 48.7 and 76.6 days respectively; p = .0001), the date first seen by a HNC service until first presentation at tumor board ([Formula: see text] = 25.1 and 38 days respectively; p = .001), mean total package time for patients who received surgery only ([Formula: see text] = 3.7 and 9.0 days respectively; p = .017), and mean total package time for patients who received surgery plus adjuvant CXRT ([Formula: see text] = 80.2 and 112.7 days respectively; p = .035). CONCLUSION: The time to treatment was significantly reduced during the COVID-19 pandemic as compared to pre-pandemic. This transparent model of patient-centered operative-room prioritization can serve as a model for improving resource allocation and efficiency of HNC care during emergency and non-emergency scenarios.

Using an Affordable Motion Capture System to Evaluate the Prognostic Value of Drop Vertical Jump Parameters for Noncontact ACL Injury.

BACKGROUND: Knee kinematic parameters during a drop vertical jump (DVJ) have been demonstrated to be associated with increased risk of noncontact anterior cruciate ligament (ACL) injury. However, standard motion analysis systems are not practical for routine screening. Affordable and practical motion sensor alternatives exist but require further validation in the context of ACL injury risk assessment. PURPOSE/HYPOTHESIS: To prospectively study DVJ parameters as predictors of noncontact ACL injury in collegiate athletes using an affordable motion capture system (Kinect; Microsoft). We hypothesized that athletes who sustained noncontact ACL injury would have larger initial and peak contact coronal abduction angles and smaller peak flexion angles at the knee during a DVJ. STUDY DESIGN: Case-control study; Level of evidence, 3. METHODS: 102 participants were prospectively recruited from a collegiate varsity sports program. A total of 101 of the 102 athletes (99%) were followed for an entire season for noncontact ACL injury. Each athlete performed 3 DVJs, and the data were recorded using the motion capture system. Initial coronal, peak coronal, and peak sagittal angles of the knee were identified by our software. RESULTS: Five of the 101 athletes sustained a noncontact ACL injury. Peak coronal angles were significantly greater and peak sagittal flexion angles were significantly smaller in ACL-injured athletes (P = .049, P = .049, respectively). Receiver operating characteristic (ROC) analysis demonstrated an area under the curve of 0.88, 0.92, and 0.90 for initial coronal, peak coronal, and peak sagittal angle, respectively. An initial coronal angle cutoff of 2.96° demonstrated 80% sensitivity and 72% specificity, a peak coronal angle cutoff of 6.16° demonstrated 80% sensitivity and 72% specificity, and a peak sagittal flexion cutoff of 93.82° demonstrated 80% sensitivity and 74% specificity on the study cohort. CONCLUSION: Increased peak coronal angle and decreased peak sagittal angle during a DVJ were significantly associated with increased risk for noncontact ACL injury. Based on ROC analysis, initial coronal angle showed good prognostic ability, whereas peak coronal angle and peak sagittal flexion provided excellent prognostic ability. Affordable motion capture systems show promise as cost-effective and practical options for large-scale ACL injury risk screening.

Patient frailty association with cerebral arteriovenous malformation microsurgical outcomes and development of custom risk stratification score: an analysis of 16,721 nationwide admissions.

OBJECTIVE: Patient frailty is associated with poorer perioperative outcomes for several neurosurgical procedures. However, comparative accuracy between different frailty metrics for cerebral arteriovenous malformation (AVM) outcomes is poorly understood and existing frailty metrics studied in the literature are constrained by poor specificity to neurosurgery. This aim of this paper was to compare the predictive ability of 3 frailty scores for AVM microsurgical admissions and generate a custom risk stratification score. METHODS: All adult AVM microsurgical admissions in the National (Nationwide) Inpatient Sample (2002-2017) were identified. Three frailty measures were analyzed: 5-factor modified frailty index (mFI-5; range 0-5), 11-factor modified frailty index (mFI-11; range 0-11), and Charlson Comorbidity Index (CCI) (range 0-29). Receiver operating characteristic curves were used to compare accuracy between metrics. The analyzed endpoints included in-hospital mortality, routine discharge, complications, length of stay (LOS), and hospitalization costs. Survey-weighted multivariate regression assessed frailty-outcome associations, adjusting for 13 confounders, including patient demographics, hospital characteristics, rupture status, hydrocephalus, epilepsy, and treatment modality. Subsequently, k-fold cross-validation and Akaike information criterion-based model selection were used to generate a custom 5-variable risk stratification score called the AVM-5. This score was validated in the main study population and a pseudoprospective cohort (2018-2019). RESULTS: The authors analyzed 16,271 total AVM microsurgical admissions nationwide, with 21.0% being ruptured. The mFI-5, mFI-11, and CCI were all predictive of lower rates of routine discharge disposition, increased perioperative complications, and longer LOS (all p < 0.001). Their AVM-5 risk stratification score was calculated from 5 variables: age, hydrocephalus, paralysis, diabetes, and hypertension. The AVM-5 was predictive of decreased rates of routine hospital discharge (OR 0.26, p < 0.001) and increased perioperative complications (OR 2.42, p < 0.001), postoperative LOS (+49%, p < 0.001), total LOS (+47%, p < 0.001), and hospitalization costs (+22%, p < 0.001). This score outperformed age, mFI-5, mFI-11, and CCI for both ruptured and unruptured AVMs (area under the curve [AUC] 0.78, all p < 0.001). In a pseudoprospective cohort of 2005 admissions from 2018 to 2019, the AVM-5 remained significantly associated with all outcomes except for mortality and exhibited higher accuracy than all 3 earlier scores (AUC 0.79, all p < 0.001). CONCLUSIONS: Patient frailty is predictive of poorer disposition and elevated complications, LOS, and costs for AVM microsurgical admissions. The authors' custom AVM-5 risk score outperformed age, mFI-5, mFI-11, and CCI while using threefold less variables than the CCI. This score may complement existing AVM grading scales for optimization of surgical candidates and identification of patients at risk of postoperative medical and surgical morbidity.