CRISPR/Cas9 cleavages in budding yeast reveal templated insertions and strand-specific insertion/deletion profiles.

Harnessing CRISPR-Cas9 technology provides an unprecedented ability to modify genomic loci via DNA double-strand break (DSB) induction and repair. We analyzed nonhomologous end-joining (NHEJ) repair induced by Cas9 in budding yeast and found that the orientation of binding of Cas9 and its guide RNA (gRNA) profoundly influences the pattern of insertion/deletions (indels) at the site of cleavage. A common indel created by Cas9 is a 1-bp (+1) insertion that appears to result from Cas9 creating a 1-nt 5' overhang that is filled in by a DNA polymerase and ligated. The origin of +1 insertions was investigated by using two gRNAs with PAM sequences located on opposite DNA strands but designed to cleave the same sequence. These templated +1 insertions are dependent on the X-family DNA polymerase, Pol4. Deleting Pol4 also eliminated +2 and +3 insertions, which are biased toward homonucleotide insertions. Using inverted PAM sequences, we also found significant differences in overall NHEJ efficiency and repair profiles, suggesting that the binding of the Cas9:gRNA complex influences subsequent NHEJ processing. As with events induced by the site-specific HO endonuclease, CRISPR-Cas9-mediated NHEJ repair depends on the Ku heterodimer and DNA ligase 4. Cas9 events are highly dependent on the Mre11-Rad50-Xrs2 complex, independent of Mre11's nuclease activity. Inspection of the outcomes of a large number of Cas9 cleavage events in mammalian cells reveals a similar templated origin of +1 insertions in human cells, but also a significant frequency of similarly templated +2 insertions.

Noteworthy Neurological Manifestations Associated With COVID-19 Infection.

March 11, 2020 marked the start of the coronavirus disease 2019 (COVID-19) pandemic. COVID-19, caused by severe acute respiratory syndrome coronavirus 2, was being reported as a severe respiratory illness. However, since the recognition of this novel virus, there has been a constant realization that it may present or manifest in a multitude of ways. At first, the typical signs and symptoms were what one would expect from a respiratory virus: cough, shortness of breath, and fever. However, as the disease became more prevalent, neurologic symptoms were reported such as headaches, hypogeusia, and hyposmia. This case report aims to add to the growing body of neurologic manifestations by presenting two cases, Bell's palsy and Guillain-Barre syndrome. Each case involves flaccid paralysis as the primary presentation.

Retraction: Noteworthy Neurological Manifestations Associated With COVID-19 Infection.

[This retracts the article DOI: 10.7759/cureus.8992.].

Renin-Angiotensin-Aldosterone System Inhibitors and COVID-19: A Meta-Analysis and Systematic Review.

Introduction Increased virulence, the severity of illness, and mortality have all been hypothesized with respect to angiotensin-converting enzyme inhibitor (ACEi)/angiotensin receptor blocker (ARB) use in coronavirus disease 2019 (COVID-19) infection. Our study aims to assess whether ACEi/ARB use in patients with COVID-19 conferred worsened severity of illness or increased mortality. Additionally, we explore the possibility of an unearthed protective benefit due to their interruption of the RAS signaling pathway as observed in cardiovascular diseases. Methods The Cochrane Library, MEDLINE, and EMBASE were searched for studies relevant to COVID-19 severity, mortality, and inflammation in the context of ACEi/ARB use. Eight studies were included with a total of 17,943 patients, 4,292 (23.9%) of which were taking an ACEi or an ARB. The study population was 47.9% female and the average age across all studies was 65. The studies chosen had a sample size of at least 100 patients. Results Mortality outcomes were assessed in six studies and showed no significant difference in mortality among the ACEi/ARB and control groups (odds ratio [OR]: 0.99, 95%CI: 0.48-2.04). Seven studies assessed the severity of COVID-19 and showed no statistically significant difference in disease severity when comparing the ACEi/ARB group to the control group (odds ratio [OR]: 1.30, 95% CI 0.87-1.94). Four studies reported the length of stay with no significant difference between the ACEi/ARB groups as compared to non-users. Four studies included inflammatory markers C-reactive protein (CRP) and D-Dimer, which were noted to be consistently lower in the ACEi/ARB groups when compared to control groups, however, this was not statistically significant. Conclusion Our study found no significant difference in mortality, severity of illness, or length of stay between ACEi/ARB users and non-users with COVID-19 infection. These results support the continuation of ACEi and ARBs in the setting of COVID-19 as advised by the American College of Cardiology (ACC)/American Heart Association (AHA). The decrease in CRP and D-dimer suggests a possible protective effect related to ACEi/ARB use in COVID-19, however, more studies with larger sample sizes are needed to establish this effect.

A Diagnostically Challenging Parotid Gland Tumor With Hybrid Features.

Salivary gland tumors are relatively uncommon with most being benign. When diagnosed the most common benign and malignant tumors are pleomorphic adenoma and mucoepidermoid carcinoma (MEC), respectively. However, not uncommonly, it is difficult to differentiate between the histopathological entities, leading to a diagnostic dilemma that can impact a patient's treatment and prognosis. A 24-year-old woman presented with a three-year history of asymptomatic left-sided facial swelling. She denied any prior history of head and neck radiation. There was no history of alcohol consumption or smoking exposure and there was no personal or family history of head and neck cancers. Additionally, she did not have any known occupational or environmental exposures. Due to the chronicity and painless nature of this facial mass, our patient did not pursue evaluation initially. Subsequently, she experienced an increase in size and pain for a few months exacerbated by swallowing. She had no other symptoms. On physical examination, a 3 x 3 cm left parotid gland mass was noted. There was no associated head or neck lymphadenopathy and compression of the left facial mass did not elicit secretions from the opening of Stensen's duct. Due to the rapid increase in size, she was sent for CT neck/soft tissue with contrast which confirmed a 3.56 x 2.67 cm solid nodule within the superficial portion of the left parotid gland. This was followed by an MRI orbit/face/neck with and without contrast, for further delineation, which demonstrated a 4 x 3.7 x 3 cm complex heterogeneous mass within the superficial left parotid gland. Thereafter the patient underwent an uncomplicated ultrasound-guided biopsy of the parotid mass. The histopathological appraisal concluded that this was a cellular pleomorphic adenoma, with mucinous and squamous metaplasia with reactive lymph nodes. Due to the new rapid increase in size and intense painful nature of this tumor, nerve-sparing left parotidectomy, fat grafting and reconstruction were completed. Cellular pleomorphic adenomas are benign low-grade neoplasms, typified as biphasic with both epithelial and myoepithelial components. However, they have increased cellularity and focally increased mitotic activity, not advanced enough to qualify as malignant. The presence of mucinous and squamous metaplasia is of diagnostic interest as it makes diagnosis on fine-needle aspiration (FNA) morphologically challenging. These findings are typical of MEC and on FNA can be misleading in the setting of a pleomorphic adenoma. However, on histopathological evaluation of the gross specimen along with immunohistochemical staining the diagnosis is made much easier. A diagnosis of MEC would have potentially required neck dissection and adjuvant therapy with a potential increased risk of morbidity and mortality. This case emphasises the importance of an adequate tissue biopsy in regards to parotid gland tumors to optimise a patient's care plan.

Transient Cardiac Dysfunction Following a Cerebrovascular Accident.

Acute cerebral injuries are often accompanied by sudden electrocardiogram (ECG) changes such as cardiac arrhythmias, QT prolongation, and abnormal T-wave morphology. One rare phenomenon is "cerebral T-waves", which are T-waves observed in the context of stroke and described as transient, symmetric, and deeply inverted. The classic cerebral T wave is defined as a T-wave inversion of ≥5 mm depth in at least four contiguous precordial leads, and it is more commonly observed in the setting of acute ischemic stroke rather than hemorrhagic stroke. We describe the case of a patient who initially presented with acute pulmonary edema, T-wave inversions in the precordial leads, and left ventricular dysfunction on echocardiogram raising suspicion of an ischemic cardiac event. However, a brain CT scan performed on the third day of admission proved us wrong.

Retraction: Transient Cardiac Dysfunction in Cerebrovascular Accidents.

[This retracts the article DOI: 10.7759/cureus.10185.].

Association Between COVID-19 Outcomes and Patient Characteristics: A Study in an Inner-City Community Hospital.

Background It has been shown that certain hematological conditions, such as lymphopenia and thrombocytopenia, are associated with increased severity and mortality from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. However, a majority of the previous data came from large institutional centers with high academic output. We aimed to explore the association between patient's characteristics, hematological parameters, and outcomes in admitted persons with coronavirus disease 2019 (COVID-19) at our community hospital in an inner city. Methods This study is a retrospective chart review designed to evaluate the potential associations between demographic and clinical characteristics of our patient population and their outcomes when testing positive for SARS-CoV-2. The study population included patients hospitalized in the Saint Francis Medical Center from January 2020 to September 2020. This pilot study included 50 out of the 275 hospitalized patients with a confirmed SARS-CoV-2 infection during this timeframe. Data collection from the patient's chart included age, sex, comorbidities, admission complete blood cell count, and use of Remdesivir, steroids, and plasma. The primary outcome was all-cause in-hospital mortality. Secondary outcomes included the need for mechanical ventilation and ICU admission. Results In this pilot study, there was an overall mortality rate of 32% (16 out of 50 patients). Charlson Comorbidity Index (CCI) of 3 points or above was present in 87.5% of the patients in the mortality group versus 41.1% in patients who survived (p = 0.0021). There was no statistically significant difference in mortality between males and females after adjusting for other variables with an odds ratio (OR) of 0.19 (95% CI 0.02-1.80, p = 0.09). There were no statically significant differences in mortality between Caucasians, non-Hispanic, Black, and Latinx patients (p = 0.466). Admission platelets were lower in the mortality group with a mean of 157.7 ± 43.23 (Thou/ul) versus 250.06 ± 93.95 (Thou/ul) in the survivors (p = 0.0005). Admission white blood cell count in the mortality group was lower than the survivor group with an average of 5.93 ± 2.58 (Thou/ul) versus 9.3 ± 4.14 (Thou/ul) (p = 0.0039), respectively. The plasma D-dimer level of 3 mg/L fibrinogen equivalent unit (FEU) or higher was associated with increased mortality. There was no association of C-reactive protein (CRP) with mortality (p = 0.93 and p = 0.54, respectively); however, the CRP level revealed an association with ICU admission (p = 0.03). The use of steroids, Remdesivir, and plasma did not have a statistically significant effect on mortality, ICU admission, or sepsis in our study. Conclusion In this study, older age, higher CCI, and plasma D-dimer level of 3 mg/L FEU or higher were associated with higher mortality among COVID-19 patients. White blood cell count and platelet count were significantly lower in the mortality group in comparison to the survivor group. However, there was no statistical difference in lymphocyte count between the mortality group and the survivor group. COVID-19 patients with thrombocytopenia or serum CRP level of 15 mg/dL or higher were more likely to be admitted to ICU.

Noteworthy Neurological Manifestations Associated With COVID-19 Infection.

March 11, 2020, marked the start of the coronavirus disease 2019 (COVID-19) pandemic. COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was being reported as severe respiratory illness. However, since the recognition of this novel virus, there has been a constant realization that it may present or manifest in a multitude of ways. At first, the typical signs and symptoms were what one would expect from a respiratory virus: cough, shortness of breath, and fever. However, as the disease became more prevalent, neurologic symptoms were reported such as headaches, hypogeusia, and hyposmia. This case report aims to add to the growing body of neurologic manifestations by presenting two cases, Bell's palsy and Guillain-Barre Syndrome. Each case involves flaccid paralysis as the primary presentation.

Transient Cardiac Dysfunction in Cerebrovascular Accidents.

Acute cerebral injuries have been repeatedly correlated with sudden and different electrocardiogram (EKG) changes, such as cardiac arrhythmias, QT prolongation, and abnormal T-wave morphology. One rare phenomenon is "cerebral T-waves," which are T-waves observed in the context of stroke and described as transient, symmetric, and deeply inverted. Moreover, few studies linked acute cerebral injuries with transient cardiac dysfunction secondary to autonomic dysfunction. The classic cerebral T-waves are defined as a T-wave inversion of ≥5 mm depth in ≥4 contiguous precordial leads, and it is more commonly observed in the setting of acute ischemic stroke rather than hemorrhagic stroke. We present a patient who presented with acute pulmonary edema, T-wave inversions in the precordial leads, and left ventricular (LV) dysfunction initially suspicious for acute coronary syndrome (ACS). However, computer tomography of the brain performed on the third day of his hospital stay proved us wrong.

Diagnosing Meat Allergy After Tick Bite Without Delay.

Galactose-α-1, 3 galactose (α-gal) is a carbohydrate found in mammalian meat. In 2007, it was implicated as a cause of severe hypersensitivity reactions when a study found elevated levels of antibodies directed against this oligosaccharide among patients treated with cetuximab, a monoclonal antibody that contained an α -gal epitope. The majority of these cases were reported in the Southeast United States in a distribution similar to that of Rocky Mountain spotted fever and ehrlichiosis, and that geographic association led researchers to the conclusion that a bite from the Lone Star tick can induce this antibody. Here, we present a case of delayed urticarial angioedema due to a mammalian meat allergy caused by α-gal immunoglobulin E acquired after tick exposures, and the knowledge and patient education required to prevent recurrences. It is estimated that approximately 0.5% to 1.0% of the general population will experience an episode of angioedema in their lifetime, and this case demonstrates why clinicians in areas that are inhabited by ticks, particularly the Lone Star species, should include this cause in their differential.