scholarly journals Lessening of porcine epidemic diarrhoea virus susceptibility in piglets after editing of the CMP-N-glycolylneuraminic acid hydroxylase gene with CRISPR/Cas9 to nullify N-glycolylneuraminic acid expression

2018 ◽  
Author(s):  
Ching-Fu Tu ◽  
Chin-kai Chuang ◽  
Kai-Hsuan Hsiao ◽  
Chien-Hong Chen ◽  
Chie-Min Chen ◽  
...  
Keyword(s):  
Gene Editing ◽  
Clinical Symptoms ◽  
Glucose Solution ◽  
Clinical Manifestations ◽  
Immunofluorescence Staining ◽  
Wild Type ◽  
Guide Rnas ◽  
Porcine Epidemic Diarrhoea Virus ◽  
Diarrhoea Virus ◽  
Cas9 Mrna

Porcine epidemic diarrhoea virus (PEDV) devastates the health of piglets but may not infect piglets whose CMP-N-glycolylneuraminic acid hydroxylase (CMAH) gene mutated (knockouts, KO) by using CRISPR/Cas9 gene editing techniques. This hypothesis was tested by using KO piglets challenged with PEDV. Two single-guide RNAs targeting the CMAH gene and Cas9 mRNA were microinjected into the cytoplasm of newly fertilized eggs; 4 live founders generated and proven to be biallelic KO, lacking detectable N-glycolylneuraminic acid (NGNA). The founders were bred, and homozygous offspring were obtained. Two-day-old (in exps. I and III) and 3-day-old (in exp. II) KO and wild-type (WT) piglets were inoculated with TCID50 1x103 PEDV and then fed 20 mL of infant formula (in exps. I and II) or sow’s colostrum (in exp. III) every 4 hours. In exp. III, the colostrum was offered 6 times and was then replaced with Ringer/5% glucose solution. At 72 hours post-PEDV inoculation (hpi), the animals were euthanized for necropsy, and their intestines were sampled. In all 3 experiments, the piglets showed apparent outward clinical manifestations suggesting that infection occurred despite the CMAH KO. In exp. I, all 6 WT piglets and only 1 of 6 KO piglets died at 72 hpi. Histopathology and immunofluorescence staining showed that the villus epithelial cells of WT piglets were severely exfoliated, but only moderate exfoliation and enterocyte vacuolization was observed in KO piglets. In exp. II, delayed clinical symptoms appeared, yet the immunofluorescence staining/histopathologic inspection (I/H) scores of the two groups differed little. In exp. III, the animals exhibited clinical and pathological signs after inoculation similar to those in exp. II. These results suggest that porcine CMAH KO with nullified NGNA expression are not immune to PEDV but that this KO may lessen the severity of the infection and delay its occurrence.

scholarly journals Correction of RNA splicing defect in β654-thalassemia mice using CRISPR/Cas9 gene-editing technology

Haematologica ◽  
2021 ◽  
Author(s):  
Dan Lu ◽  
Xiuli Gong ◽  
Yudan Fang ◽  
Xinbing Guo ◽  
Yanwen Chen ◽  
...  
Keyword(s):  
Survival Rate ◽  
Genome Editing ◽  
Molecular Mechanism ◽  
Splice Site ◽  
Rna Splicing ◽  
Gene Editing ◽  
Clinical Symptoms ◽  
Rna Expression ◽  
Wild Type ◽  
Acceptor Splice Site

β654-thalassemia is a prominent Chinese subtype of β-thalassemia, representing 17% of total β-thalassemia cases in China. The molecular mechanism underlying this subtype involves the IVS-2-654 C→T mutation leading to aberrant β-globin RNA splicing. This results in an additional 73-nucleotide exon between exons 2 and 3 and leads to severe thalassemia syndrome. Herein, we explored a CRISPR/Cas9 genome editing approach to eliminate the additional 73-nt by targeting both the IVS-2-654 C→T and a cryptic acceptor splice site at IVS-2-579 in order to correct aberrant β-globin RNA splicing and ameliorate the clinical β-thalassemia syndrome in β654 mice. Gene-edited mice were generated by microinjection of sgRNAs and Cas9 mRNAs into 1-cell embryos of β654 or control mice. 83.3% of live-born mice were gene-edited, 70% of which produced correctly spliced RNA. No off-target events were observed. The clinical symptoms, including hematologic parameters and tissue pathology of all of the edited-β654 founders and their offspring, were significantly improved compared to the non-edited β654 mice, consistent with the restoration of wild-type β-globin RNA expression. Notably, the survival rate of gene-edited heterozygous β654 mice increased significantly, and live-born homozygous β654 mice were observed. Our study demonstrated a new and effective gene-editing approach that may provide a groundwork for the exploration of β654-thalassemia therapy in the future.

scholarly journals High-Throughput Profiling of Cas12a Orthologues and Engineered Variants for Enhanced Genome Editing Activity

2021 ◽  
Vol 22 (24) ◽  
pp. 13301
Author(s):  
Dan Zhu ◽  
Junyi Wang ◽  
Di Yang ◽  
Jianzhong Xi ◽  
Juan Li
Keyword(s):  
High Throughput ◽  
Gene Editing ◽  
Genome Engineering ◽  
Human Cells ◽  
Wild Type ◽  
Guide Rnas ◽  
Promising Tool ◽  
Target Sites ◽  
Editing Activity ◽  
Type V

CRISPR/Cas12a (formerly Cpf1), an RNA-guided endonuclease of the Class II Type V-A CRISPR system, provides a promising tool for genome engineering. Over 10 Cas12a orthologues have been identified and employed for gene editing in human cells. However, the functional diversity among emerging Cas12a orthologues remains poorly explored. Here, we report a high-throughput comparative profiling of editing activities across 16 Cas12a orthologues in human cells by constructing genome-integrated, self-cleaving, paired crRNA–target libraries containing >40,000 guide RNAs. Three Cas12a candidates exhibited promising potential owing to their compact structures and editing efficiency comparable with those of AsCas12a and LbCas12a, which are well characterized. We generated three arginine substitution variants (3Rv) via structure-guided protein engineering: BsCas12a-3Rv (K155R/N512R/K518R), PrCas12a-3Rv (E162R/N519R/K525R), and Mb3Cas12a-3Rv (D180R/N581R/K587R). All three Cas12a variants showed enhanced editing activities and expanded targeting ranges (NTTV, NTCV, and TRTV) compared with the wild-type Cas12a effectors. The base preference analysis among the three Cas12a variants revealed that PrCas12a-3Rv shows the highest activity at target sites with canonical PAM TTTV and non-canonical PAM TTCV, while Mb3Cas12a-3Rv exhibits recognition features distinct from the others by accommodating for more nucleotide A at position −3 for PAM TATV and at position −4 for PAM ATCV. Thus, the expanded Cas12a toolbox and an improved understanding of Cas12a activities should facilitate their use in genome engineering.

Etiology and clinical features of epididymo-orchitis: A single-center study in Tehran, Iran

2020 ◽  
Vol 20 ◽  
Author(s):  
Sara Abolghasemi ◽  
Mohammad Alizadeh ◽  
Ali Hashemi ◽  
Shabnam Tehrani
Keyword(s):  
Chlamydia Trachomatis ◽  
Clinical Symptoms ◽  
Urine Culture ◽  
Clinical Manifestations ◽  
Urinary Frequency ◽  
Serological Tests ◽  
Duration Of Symptoms ◽  
Two Samples ◽  
History Of ◽  
Tract Infections

Introduction: Epididymo-orchitis is a common urological disease among men. Little is known about the clinical and epidemiological aspects of the disease in Iran. Thus, the present study was aimed to investigate the etiology, clinical sequelae and risk factors of patients with epididymo-orchitis in Tehran, Iran. Materials and Methods: Patients presenting with epididymo-orchitis were prospectively analyzed in order to study the etiology and pattern of the disease. Bacteriological, molecular and serological tests were undertaken to look for Chlamydia trachomatis, Neisseria gonorrhoeae, Brucella spp., Mycoplasma spp, and other bacteria. Results: Fifty patients with epididymo-orchitis were evaluated according to their clinical symptoms, duration of symptoms, physical examination, and laboratory studies. The mean age of the patients was 53 years. Fever, dysuria, pain in the flanks, urinary frequency and discharges occurred in 58.0%, 50.0%, 50.0%, 28.0% and 6.0%, respectively. Bacterial pathogen was identified in 26% (13/50) of patients by urine culture. Escherichia coli was the etiological agent in 11/13 patients (84.6%). Two out of 50 patients (4.0%) were also positive for Chlamydia trachomatis. Two samples were serologically positive for Brucella spp. High Mean age, fever, urinary frequency, history of the underlying disease and history of urinary tract infections were found to have a significant association with the positive bacteriologic urine culture (P<0.05). Conclusions: The most common clinical manifestations were fever, dysuria, and abdominal pain. E. coli and C. trachomatis were the major causative agents. Use of a set of diagnostic approaches including clinical symptoms, urine culture and more precise techniques such as PCR should be taken into consideration for the definitive diagnosis.

Scrub typhus (Orientia tsutsugamushi), A rapidly spreading epidemic in Chennai - A standalone lab experience

2016 ◽  
Vol 5 (09) ◽  
pp. 4896
Author(s):  
Sripriya C.S.* ◽  
Shanthi B. ◽  
Arockia Doss S. ◽  
Antonie Raj I. ◽  
Mohana Priya
Keyword(s):  
Scrub Typhus ◽  
Clinical Symptoms ◽  
Clinical Manifestations ◽  
Febrile Illness ◽  
Orientia Tsutsugamushi ◽  
The Past ◽  
Igm Elisa ◽  
The Mean ◽  
Specific Symptoms ◽  
Symptoms And Signs

Scrub typhus (Orientia tsutsugamushi), is a strict intracellular bacterium which is reported to be a recent threat to parts of southern India. There is re-emergence of scrub typhus during the past few years in Chennai. Scrub typhus is an acute febrile illness which generally causes non-specific symptoms and signs. The clinical manifestations of this disease range from sub-clinical disease to organ failure to fatal disease. This study documents our laboratory experience in diagnosis of scrub typhus in patients with fever and suspected clinical symptoms of scrub typhus infection for a period of two years from April 2014 to April 2016 using immunochromatography and IgM ELISA methods. The study was conducted on 648 patients out of whom 188 patients were found to be positive for scrub typhus. Results also showed that pediatric (0 -12 years) and young adults (20 – 39 years) were more exposed to scrub typhus infection and female patients were more infected compared to male. The study also showed that the rate of infection was higher between September to February which also suggested that the infection rate is proportional to the climatic condition. Statistical analysis showed that the mean age of the patients in this study was 37.6, standard deviation was 18.97, CV % was 50.45. 

scholarly journals Pediatric COVID-19 and the Factors That May Mitigate Its Clinical Course

2020 ◽  
Vol 10 (01) ◽  
pp. e137-e140
Author(s):  
Mosaad Abdel-Aziz ◽  
Nada M. Abdel-Aziz ◽  
Dina M. Abdel-Aziz ◽  
Noha Azab
Keyword(s):  
Clinical Features ◽  
Clinical Symptoms ◽  
Clinical Course ◽  
Clinical Manifestations ◽  
Gastrointestinal Symptoms ◽  
Angiotensin Converting Enzyme 2 ◽  
Compulsory Vaccination ◽  
Specific Factors ◽  
Novel Coronavirus ◽  
Radiographic Data

AbstractThe clinical manifestations of novel coronavirus disease 2019 (COVID-19) vary from mild flu-like symptoms to severe fatal pneumonia. However, children with COVID-19 may be asymptomatic or may have mild clinical symptoms. The aim of this study was to investigate clinical features of pediatric COVID-19 and to search for the factors that may mitigate the disease course. We reviewed the literature to realize the clinical features, laboratory, and radiographic data that may be diagnostic for COVID-19 among children. Also, we studied the factors that may affect the clinical course of the disease. Fever, dry cough, and fatigue are the main symptoms of pediatric COVID-19, sometimes flu-like symptoms and/or gastrointestinal symptoms may be present. Although some infected children may be asymptomatic, a recent unusual hyperinflammatory reaction with overlapping features of Kawasaki's disease and toxic shock syndrome in pediatric COVID-19 has been occasionally reported. Severe acute respiratory syndrome-coronvirus-2 (SARS-CoV-2) nucleic acid testing is the corner-stone method for the diagnosis of COVID-19. Lymphocyte count and other inflammatory markers are not essentially diagnostic; however, chest computed tomography is highly specific. Factors that may mitigate the severity of pediatric COVID-19 are home confinement with limited children activity, trained immunity caused by compulsory vaccination, the response of the angiotensin-converting enzyme 2 receptors in children is not the same as in adults, and that children are less likely to have comorbidities. As infected children may be asymptomatic or may have only mild respiratory and/or gastrointestinal symptoms that might be missed, all children for families who have a member diagnosed with COVID-19 should be investigated.

scholarly journals Gastric amyloidosis presenting as acute upper gastrointestinal bleeding: a case report

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Rachael Chan ◽  
Stephanie Carpentier
Keyword(s):  
Multiple Myeloma ◽  
Clinical Symptoms ◽  
Clinical Manifestations ◽  
Left Gastric Artery ◽  
Bleeding Complications ◽  
Al Amyloidosis ◽  
Gi Bleeding ◽  
Reduced Ejection Fraction ◽  
Repeat Endoscopy ◽  
Tertiary Center

Abstract Background Amyloidosis is characterized by extracellular tissue deposition of fibrils, composed of insoluble low-molecular-weight protein subunits. The type, location, and extent of fibril deposition generates variable clinical manifestations. Gastrointestinal (GI) bleeding due to amyloid deposition is infrequent. Previous literature describes upper GI bleeding (UGIB) in patients with known amyloid disease. Here, we describe a case of recurrent UGIB that ultimately led to a diagnosis of GI amyloidosis and multiple myeloma in a patient with no history of either. Case presentation A 76-year-old male presented to the emergency department with frank hematemesis, melena, and a decreased level of consciousness. Management required intensive care unit (ICU) admission with transfusion, intubation, and hemodynamic support. Upper endoscopy revealed gastritis with erosions and nodularity in the gastric cardia and antrum. Hemostasis of a suspected bleeding fundic varix could not be achieved. Subsequently, the patient underwent computerized tomography (CT) angiography and an interventional radiologist completed embolization of the left gastric artery to address potentially life-threatening bleeding. Complications included development of bilateral pleural effusions and subsegmental pulmonary emboli. Pleural fluid was negative for malignancy. He was transferred to a peripheral hospital for continued care and rehabilitation. Unfortunately, he began re-bleeding and was transferred back to our tertiary center, requiring re-admission to the ICU and repeat endoscopy. Repeat biopsy of the gastric cardial nodularity was reported as active chronic gastritis and ulceration. However, based on the unusual endoscopic appearance, clinical suspicion for malignancy remained high. He exhibited symptoms of congestive heart failure following standard resuscitation. Transthoracic echocardiogram (TTE) demonstrated a reduced ejection fraction of 35–40% and a strain pattern with apical sparing. Following discussions between the treating gastroenterologist, consulting cardiologist, and pathologist, Congo Red staining was performed, revealing submucosal amyloid deposits. Hematology was consulted and investigations led to diagnosis of multiple myeloma (MM) and immunoglobulin light-chain (AL) amyloidosis. The patient was treated for MM for four months prior to cessation of therapy due to functional and cognitive decline. Conclusions GI amyloidosis can present with various non-specific clinical symptoms and endoscopic findings, rendering diagnosis a challenge. This case illustrates GI amyloidosis as a potential—albeit rare—etiology of UGIB.

scholarly journals CD4+ CD25+ Foxp3+ Regulatory T Cells, Dendritic Cells, and Circulating Cytokines in Uncomplicated Malaria: Do Different Parasite Species Elicit Similar Host Responses?

2010 ◽  
Vol 78 (11) ◽  
pp. 4763-4772 ◽  
Author(s):  
Raquel M. Gonçalves ◽  
Karina C. Salmazi ◽  
Bianca A. N. Santos ◽  
Melissa S. Bastos ◽  
Sandra C. Rocha ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Parasite Species ◽  
Inflammatory Responses ◽  
Clinical Symptoms ◽  
Clinical Manifestations ◽  
Surface Expression ◽  
Interleukin 10 ◽  
Treg Cells ◽  
Experimental Models ◽  
Necrosis Factor Alpha

ABSTRACT Clearing blood-stage malaria parasites without inducing major host pathology requires a finely tuned balance between pro- and anti-inflammatory responses. The interplay between regulatory T (Treg) cells and dendritic cells (DCs) is one of the key determinants of this balance. Although experimental models have revealed various patterns of Treg cell expansion, DC maturation, and cytokine production according to the infecting malaria parasite species, no studies have compared all of these parameters in human infections with Plasmodium falciparum and P. vivax in the same setting of endemicity. Here we show that during uncomplicated acute malaria, both species induced a significant expansion of CD4+ CD25+ Foxp3+ Treg cells expressing the key immunomodulatory molecule CTLA-4 and a significant increase in the proportion of DCs that were plasmacytoid (CD123+), with a decrease in the myeloid/plasmacytoid DC ratio. These changes were proportional to parasite loads but correlated neither with the intensity of clinical symptoms nor with circulating cytokine levels. One-third of P. vivax-infected patients, but no P. falciparum-infected subjects, showed impaired maturation of circulating DCs, with low surface expression of CD86. Although vivax malaria patients overall had a less inflammatory cytokine response, with a higher interleukin-10 (IL-10)/tumor necrosis factor alpha (TNF-α) ratio, this finding did not translate to milder clinical manifestations than those of falciparum malaria patients. We discuss the potential implications of these findings for species-specific pathogenesis and long-lasting protective immunity to malaria.

scholarly journals Clinical Manifestations of Hemochromatosis inHFEC282Y Homozygotes Identified by Screening

2008 ◽  
Vol 22 (11) ◽  
pp. 923-930 ◽  
Author(s):  
Gordon D McLaren ◽  
Christine E McLaren ◽  
Paul C Adams ◽  
James C Barton ◽  
David M Reboussin ◽  
...  
Keyword(s):  
Iron Overload ◽  
Serum Ferritin ◽  
Elevated Serum ◽  
Clinical Signs ◽  
Clinical Manifestations ◽  
Joint Stiffness ◽  
Chronic Fatigue ◽  
Newly Diagnosed ◽  
Wild Type ◽  
Control Subjects

BACKGROUND: Patients with hemochromatosis may suffer organ damage from iron overload, often with serious clinical consequences.OBJECTIVE: To assess prevalences of self-reported symptoms and clinical signs and conditions in persons homozygous for the hemochromatosis gene (HFE)mutation (C282Y) identified by screening.METHODS: Participants were adults 25 years of age or older enrolled in the Hemochromatosis and Iron Overload Screening (HEIRS) Study. C282Y homozygotes (n=282) were compared with control participants without theHFEC282Y or H63D alleles (ie, wild type/wild type; n=364).RESULTS: Previously diagnosed C282Y homozygotes and newly diagnosed homozygotes with elevated serum ferritin levels had higher prevalences of certain symptoms such as chronic fatigue (OR 2.8; 95% CI 1.34 to 5.95, and OR 2.0; 95% CI 1.07 to 3.75, respectively), and had more hyperpigmentation on physical examination (OR 4.7; 95% CI 1.50 to 15.06, and OR 3.7; 95% CI 1.10 to 12.16, respectively) and swelling or tenderness of the second and third metacarpophalangeal joints (OR 4.2; 95% CI 1.37 to 13.03, and OR 3.3; 95% CI 1.17 to 9.49, respectively) than control subjects. Joint stiffness was also more common among newly diagnosed C282Y homozygotes with elevated serum ferritin than among control subjects (OR 2.7; 95% CI 1.38 to 5.30). However, the sex- and age-adjusted prevalences of self-reported symptoms and signs of liver disease, heart disease, diabetes and most other major clinical manifestations of hemochromatosis were similar in C282Y homozygotes and control subjects.CONCLUSIONS: Some symptoms and conditions associated with hemochromatosis were more prevalent among C282Y homozygotes identified by screening than among control subjects, but prevalences of most outcomes were similar in C282Y homozygotes and controls in this primary care-based study.

CLINIC AND COMPLICATIONS OF COVID-19 CORONAVIRUS DISEASE WITH PATHOGENESIS ELEMENTS

2020 ◽  
pp. 72-79
Author(s):  
I. V. Аndrusovich
Keyword(s):  
Acute Respiratory Distress Syndrome ◽  
Respiratory Tract ◽  
Respiratory Distress Syndrome ◽  
Respiratory Distress ◽  
Distress Syndrome ◽  
Clinical Symptoms ◽  
Clinical Course ◽  
Clinical Manifestations ◽  
Second Phase ◽  
Upper Respiratory Tract

The longer the COVID−19 coronavirus pandemic lasts, the more information about its clinical manifestations is accumulated. The incubation period of COVID−19 ranges from 2 to 14 days, rarely up to 3 weeks, but in a significant number of cases an infection is not accompanied with the appearance of clinical symptoms. Currently, the following variants of the clinical course of COVID−19 can be identified as follows: viral load; subclinical; slight; uncomplicated with damage to only the upper respiratory tract; mild pneumonia, severe pneumonia, acute respiratory distress syndrome, etc. The clinical course of COVID−19 depends on the severity, the criteria of which are the intoxication manifestation, the degree of fever and the dominant syndrome. Mild / moderate forms are manifested by frequent increase in body temperature up to 38 ° C, respiratory symptoms, headache, myalgias, palpitations and general malaise. Patients stop distinguishing smells and feel the taste of food. Approximately from the 7th to the 9th days of the disease there are problems with breathing, which indicates the impairment of the lower respiratory tract and the beginning of the second phase of the disease, and its course is regarded as severe. Severe forms of the disease can also be manifested by impaired coordination of movements, slurred speech. In 1 to 4 % of patients there is developed the psychosis in the form of hallucinations. In the elderly, COVID−19 may be accompanied by delirium, lowering blood pressure. The risks of adverse disease are associated with somatic diseases: cardiovascular and nervous systems, respiratory tract, hormonal disorders, etc. Otitis, sinusitis, sepsis, bronchopulmonary infection, thrombosis, myocarditis etc. can be the complications of COVID−19. Computer tomography is an instrumental test that demonstrates the damage of lungs with coronavirus and allows to assess its severity. Key words: coronavirus infection, COVID−19, clinical variants, severity, pneumonia, acute respiratory distress syndrome.

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