Carrier State Research Articles

P104: BASE EDITING REPAIRS THE HBE MUTATION RESTORING THE PRODUCTION OF NORMAL GLOBIN CHAINS IN SEVERE HBE/β-THALASSEMIA PATIENT HEMATOPOIETIC STEM AND ERYTHROID CELLS

Aims: HbE/β-thalassemia is the commonest form of severe β-thalassemia, and comprises approximately 50% of all cases worldwide1. HbE/β-thalassemia is caused by the HbE codon 26 G>A mutation on one allele and any β0-thalassemia mutation on the other. There is a reduction in β-globin production, resulting in a relative excess in α-globin chains that leads to ineffective erythropoiesis. Importantly, individuals with a mutation on one, but not two, alleles have β-thalassemia trait, a carrier state with a normal phenotype shared by 1.5% of the world’s population2. Recent gene therapy and gene editing approaches have been developed to treat β-thalassemia but do not directly repair the causative mutation in-situ. Gene replacement approaches rely on lentiviral vector-based sequence insertion or homology directed repair (HDR). HbF induction strategies rely on non-homologous end joining targeting of enhancers in-trans. These approaches, whilst variably successful, are associated with potential safety concerns. Methods: Adenine base editors (ABEs) circumvent these problems by directly repairing pathogenic variants in-situ through deamination. ABEs catalyse A-T to G-C transitions. Conversion of the HbE codon to WT through base editing is an attractive strategy to recapitulate the phenotypically normal β-thalassemia trait state without potentially harmful double-strand breaks or random vector insertions. ABEs are able to convert the HbE codon (AAG) to wild-type (GAG), but also to GGG or AGG (Fig A). GGG at codon 26 is found in a naturally occurring haemoglobin, Hb Aubenas3. Heterozygotes have normal red cell indices and are phenotypically normal. We electroporated the latest generation of ABE8 editors4 as mRNA into 3 different severe HbE/β-thalassemia donor HSPCs with sgRNAs targeting the HbE codon. Results: The mean conversion from the HbE codon to a normal or normal variant in unselected cells was 86.2 (SD±8.1%, Fig B). The indel rate from inadvertent on-target Cas9 cleavage was below 0.5%. Edited cells did not show any perturbations in erythroid differentiation as assessed by Immunophenotyping and cellular morphology. In differentiated erythroid cells, RT-qPCR showed a mean fall in the α/β mRNA ratio to 0.65±0.08 (unedited patient cells normalised to 1, n=5, Figure C), indicating a reduction in the excess α-globin gene expression. Protein analysis by CE-HPLC showed a 3.6-fold reduction in HbE levels (SD±1.3) and a 13.5-fold increase in HbA/Hb Aubenas (SD±2.4, Fig D & E). In serial NSG-murine xenotransplantation experiments, base edited cells were found to persist in secondary transplants, showing editing is possible in long-term HSCs (mean editing efficiency 34.5%, Fig F). Potential off-target effects were assessed in-vitro by CIRCLE-seq5; most candidate sites were in intergenic and intronic regions (Fig G & H). The top 250 sites were sequenced using deep targeted NGS. Only 5 sites showed OT deaminations at low levels in patient cells (mean 1.5%). We developed a machine learning model to assess potential OT-effects on chromatin accessibility, at all candidate sites in 49 different blood cell types6. Only 17 potential edits were predicted to result in a significant change in chromatin state (Fig I). 3 of these were in the top 250 sites sequenced previously, and none showed deamination in-vivo. Conclusion: Together these data provide robust evidence for base editing being used as an effective and safe therapeutic strategy for HbE/β-thalassemia.

Effect of inserted Al<sub><i>x</i></sub>Ga<sub>1–<i>x</i></sub>N layer on characteristic of double-channel <i>n</i>-Al<sub>0.3</sub>Ga<sub>0.7</sub>N/GaN/<i>i</i>-Al<sub><i>x</i></sub>Ga<sub>1–<i>x</i></sub>N/GaN HEMT

<sec>With the demand for high-temperature, high-frequency, and high-power microwave applications increasing, AlGaN/GaN high electron mobility transistors (HEMT) have attracted much attention in recent years. Two-dimensional electron gas (2DEG) induced by spontaneous polarization and piezoelectric polarization caused by the uneven charge distribution on Ga-N bond and the large tensile strain guarantees the high performance of AlGaN/GaN HEMT. Compared with single-channel devices, dual-channel AlGaN/GaN HEMT has great application prospects in enhancing the electronic confinement, current drive and alleviating the current collapse. In order to study the physical characteristics, the carrier state and transportation characterization of <i>n</i>-Al<sub>0.3</sub>Ga<sub>0.7</sub>N/GaN/i-Al<i><sub>x</sub></i>Ga<sub>1–<i>x</i></sub>N/GaN multilayer structure are investigated. By calculating the one-dimensional self-consistent Poisson-Schrödinger, the energy band diagram, electric field and charge distribution in the devices are obtained. The 2DEG, alloy disorder and dislocation scattering mechanism in the device are also analyzed by analytical models in which the wave function in finite barriers and Fermi’s rule are used.</sec><sec>With Al<i><sub>x</sub></i>Ga<sub>1–<i>x</i></sub>N layer thickness increasing from 0 nm to 30 nm and Al content rising from 0.1 to 0.2, the concentration of 2DEG localized in the heterointerface is diminished in the first channel. Simultaneously, mobility limited by alloy disorder scattering increases monotonically with the <i>r</i> composition occupation number and the Al<i><sub>x</sub></i>Ga<sub>1–<i>x</i></sub>N thickness proportion increasing. Besides, dislocation scattering on carriers is strengthened in the same quantum well, resulting in the lower mobility. In the second channel, 2DEG density gets growing when the variables mentioned above is enlarged. The mobility restricted by alloy disorder scattering shows a reverse trend with the variation of the Al<i><sub>x</sub></i>Ga<sub>1–<i>x</i></sub>N thickness and Al fraction, which more greatly affect the carriers in the parasitic channel due to the lower barrier height and high permeable carriers. Furthermore, the effect of dislocation scattering on channel electrons is gradually weakened, resulting in an increasing mobility. In general, The dislocation scattering effect in the second channel is intenser than that in the first channel.</sec>

Optimizing the quasi-equilibrium state of hot carriers in all-inorganic lead halide perovskite nanocrystals through Mn doping: fundamental dynamics and device perspectives.

Hot carrier (HC) cooling accounts for the significant energy loss in lead halide perovskite (LHP) solar cells. Here, we study HC relaxation dynamics in Mn-doped LHP CsPbI3 nanocrystals (NCs), combining transient absorption spectroscopy and density functional theory (DFT) calculations. We demonstrate that Mn2+ doping (1) enlarges the longitudinal optical (LO)–acoustic phonon bandgap, (2) enhances the electron–LO phonon coupling strength, and (3) adds HC relaxation pathways via Mn orbitals within the bands. The spectroscopic study shows that the HC cooling process is decelerated after doping under band-edge excitation due to the dominant phonon bandgap enlargement. When the excitation photon energy is larger than the optical bandgap and the Mn2+ transition gap, the doping accelerates the cooling rate owing to the dominant effect of enhanced carrier–phonon coupling and relaxation pathways. We demonstrate that such a phenomenon is optimal for the application of hot carrier solar cells. The enhanced electron–LO phonon coupling and accelerated cooling of high-temperature hot carriers efficiently establish a high-temperature thermal quasi-equilibrium where the excessive energy of the hot carriers is transferred to heat the cold carriers. On the other hand, the enlarged phononic band-gap prevents further cooling of such a quasi-equilibrium, which facilitates the energy conversion process. Our results manifest a straightforward methodology to optimize the HC dynamics for hot carrier solar cells by element doping.

Precore/core mutations of hepatitis B virus genotype D arising in different states of infection.

Aim of the studyPrecore/core variations and liver disease progression have been suggested. In this study, we aimed to determine the frequency of precore/core mutations in hepatitis B virus (HBV)-infected patients at various clinical stages.Material and methodsIn total, 73 HBV-infected patients including 26 inactive carriers (IC), 20 chronic active (CA), and 27 patients with liver cirrhosis/hepatocellular carcinoma (C/HCC) were randomly selected. The HBV DNA was extracted from the sera and subjected to nested PCR for amplification of precore/core region. The PCR product was then sequenced by the Sanger method.ResultsThe stop codon of W28*(G1896A) was determined as the most prevalent mutation (55%) of the precore region. The comparison of groups also demonstrated that core substitutions at residues of S21, E40 and I105 (< 0.05) correlated with the development of the inactive carrier state. Furthermore, the total substitutions in Th epitopes (117-131) were significantly higher in the C/HCC group than the IC and CA groups (p = 0.001).ConclusionsOur results indicated a high frequency of W28* mutation in HBV studied patients. Moreover, variations including S21, E40 and I105 and R151 that were mapped onto cellular epitopes might be related to inactive state development.

A mini-review on antimycoplasma antibiotics

Antimycoplasma drugs paved the way for the treatment of mycoplasma infections even before the vaccines were developed. Macrolides, tetracyclines, or aminoglycosides were being used for long, incorporating quinolones in the newer regimes. Former drug classes target protein synthesis, whereas the latter one targets DNA synthesis. These drugs proved quite effective and helped minimize severity and loss to farmers. However, prolonged use of antibiotics may have serious concerns of drug resistance, side effects, and drug residues in food animal products. In addition, despite treating mycoplasma infection, some drugs create a carrier state of infection that can spread the infection without being recognized. Hence, novel alternate therapeutics need to be explored to overcome these limitations whilst checking the antibiotic resistance with regular antibiotic sensitivity tests, which need to be standardized for different mycoplasma. Future therapeutics are needed under the prevailing situations of emerging and transboundary spread of mycoplasma infections.

Association of Vascular Endothelial Growth Factor 936 C/T Gene Polymorphism with Renal Allograft Outcome: A Study from North India.

The significance of vascular endothelial growth factor (VEGF) and its polymorphisms in renal allograft rejection has recently become the subject of extensive research. Recently, some studies have shown some role of VEGF in rejection episodes and graft survival. VEGF +936 C>T polymorphism is significant in the transcription regulation of VEGF. Herein, we report the results of a prospective, single-center study seeking an association of VEGF +936 C/T gene polymorphism and allograft rejection. One hundred and forty-seven kidney transplant recipients with age-and sex-matched controls were included in this study. VEGF 936 C/T genes were studied using restriction fragment length polymorphism analysis of the blood specimen of these patients. All patients were studied for allograft rejection, response to treatment, and overall graft survival. We found that CT genotype and T allele carrier state were associated with good graft outcomes (P = 0.008 and 0.002, respectively). There was a lower number of rejection episodes with T allele, although it was not a significant finding (P = 0.880). Our findings suggest that good graft outcome in kidney transplant recipients is associated with an increased frequency of the VEGF 936 CT genotype and T allele, and that determination of the T allele might be helpful for the identification of recipients with overall good graft survival.

Two In Vivo Models to Study Salmonella Asymptomatic Carrier State in Chicks.

In chicken, Salmonella Enteritidis and Salmonella Typhimurium, the two main serotypes isolated in human infections, can persist in the host organism for many weeks and up to many years without causing any symptoms. This persistence generally occurs after a short systemic infection that may either lead to death of very young birds or develop into cecal asymptomatic persistence, which is often accompanied by a high level of bacterial excretion, facilitating Salmonella transmission to counterparts. Here we describe two models of chick infection. The first model reproduces well the poultry infection in farm flocks. Numerous reinfections and animal-animal recontaminations occur leading to a high level of cecal colonization and fecal excretion in all chicks in the flock, over several weeks. In the second model, these animal reinfections and recontaminations are hampered leading to heterogeneity of infection characterized by the presence of low and super-shedders. This model allows for more mechanistic studies of Salmonella/chicks interactions as animal recontaminations are lowered.

Review on Leptospirosis and its Public Health Significance

Leptospirosis is the most widespread and most prevalent zoonotic disease caused by Leptospira bacteria this is pathogenic Gramnegative bacteria, the disease leptospirosis a zoonotic disease of global importance. It caused by various serovars of leptospira interrogans that belong to the genus Leptospira. Besides leptospira interrogans is the most serovars like the disease occurs in nearly all mammalian species. The highest burden in low-income both found in urban and in rural environments. Rodents are known as the main reservoir animals, However, all animals are reservoir include human so leptospirosis is more common in the tropical regions rather than subtropics because of the longer survival of the organism in the environment and frequent exposure of animals and humans to contaminated environments. Direct transmission to humans by broken skin and mucous membranes exposed to the urine of infected animals. While the clinical signs of leptospirosis do not vary greatly with the species of animals include laboratory tests. Control and prevention is sanitary measures, vaccination, quarantine and rodent control are the most important control measures of the disease, require elimination of the carrier state, maintenance of environmental condition and immunization of the animal, tropical areas where warm humid conditions and alkaline or neutral soil helps leptospira survive better.

Rhabdomyolysis in a hospitalized patient with COVID-19 – case report

Introduction. COVID-19 is a viral infection with a variable clinical spectrum, ranging from asymptomatic carrier state to severe pneumonia. It is associated with a variety of complications, including musculoskeletal abnormalities. Whereas myalgia is a common clinical finding at these patients, only a few cases of COVID-19-associated rhabdomyolysis have been described in the literature. Case presentation. We describe the case of a 42-year old male confirmed with SARS-CoV-2 infection who presented to the emergency department with an 11-day evolution of dyspnea, cough, fatigue, myalgia and hyperchromic urine. The physical examination revealed dyspnea and an oxygen saturation of 87% while breathing ambient air, being otherwise normal. Blood tests showed neutrophilia, increased inflammatory markers, COVID-19 associated coagulopathy and elevation of muscular enzymes creatine-kinase and myoglobin. The chest computer tomography was consistent with mixed pneumonia, distributed in all pulmonary segments and the case was interpreted as a severe form of SARS-CoV-2 infection, associated with acute respiratory failure and rhabdomyolysis. Upon treatment (Enoxaparin, Aspirin, Dexamethasone, Favipiravir, oxygen administered by face mask, fluid resuscitation), his condition considerably improved, along with the laboratory findings, and he was discharged, without developing acute kidney injury or other complications related to rhabdomyolysis during his admission. Conclusion. COVID-19 patients can develop rhabdomyolysis, which can result in life-threating complications.

Emergence of Invasive Fungal Rhinosinusitis in Recently Recovered COVID-19 Patients.

The risk of invasive fungal rhinosinusitis is increased in Coronavirus disease 2019 (COVID-19) because of its direct impact in altering innate immunity and is further exacerbated by widespread use of steroids/antibiotics/monoclonal antibodies. The study aims to describe this recently increased clinical entity in association with COVID-19. A prospective, longitudinal study including patients diagnosed with acute invasive fungal rhinosinusitis (AIFRS) who recently recovered from COVID-19 infection or after an asymptomatic carrier state. A single-center, descriptive study investigating demographic details, clinical presentation, radio-pathological aspects, and advocated management. A total of 21 patients were included with a mean age of 49.62 years (SD: 14.24). Diabetes mellitus (DM) was the most common underlying disorder (90.48%), and 63.16% of all patients with DM had a recent onset DM, either diagnosed during or after COVID-19 infection. Nineteen patients (90.48%) had recently recovered from active COVID-19 infection, and all had a history of prior steroid treatment (oral/parenteral). Remaining 2 patients were asymptomatic COVID-19 carriers. Surprisingly, 2 patients had no underlying disorder, and 5 (23.81%) recently received the Covishield vaccine. Fungal analysis exhibited Mucor (95.24%) and Aspergillus species (14.29%). Most common sign/symptom was headache and facial/periorbital pain (85.71%), followed by facial/periorbital swelling (61.90%). Disease involvement: sinonasal (100%), orbital (47.62%), pterygopalatine fossa (28.58%), infratemporal fossa (14.29%), intracranial (23.81%), and skin (9.52%). Exclusive endoscopic debridement and combined approach were utilized in 61.90% and 38.10%, respectively. Both liposomal amphotericin B and posaconazole were given in all patients except one. A high suspicion of AIFRS should be kept in patients with recent COVID-19 infection who received steroids and presenting with headache, facial pain, and/or facial swelling. Asymptomatic COVID-19 carriers and COVID-19 vaccinated candidates are also observed to develop AIFRS, although the exact immuno-pathogenesis is still unknown. Prompt diagnosis and early management are vital for a favorable outcome.

Frequency of Haemoglobinopathies in Premarital Screening in Nineveh Province

Background: Haemoglobinopathy is a large heterogeneous group of genetic abnormalities of haemoglobin. It is one of the most common inherited diseases worldwide. Aim of this study: This study aimed to find the frequency of different types of Haemoglobinopathies in premarital couples in Nineveh province. Subjects and Methods: In this cross-sectional study, the subjects were couples who go to the primary health care centers in Nineveh governorate for routine premarital investigations and the data were collected from the main premarital screening centers in Nineveh governorate. Results: In this study, 1127 cases were included. 613 (54.4 %) were male, their ages range between (13-80 years), and 514 (45.6 %) were female, their ages range between (10-52 years). 47 cases were diagnosed as βeta-thalassaemia carriers with an overall frequency of 4.2 %. Ten cases had haemoglobin S (HbS) by the High Performance Liquid Chromatography (HPLC) (sickle cell trait in 9 cases and sickle β-thalassaemia in only one case); the overall frequency of sickle cell carrier state is 0.89 %. Eight cases with an overall frequency of 0.71 % were diagnosed as having other types of Haemoglobinopathies (haemoglobin D, haemoglobin E, haemoglobin H). Eighty one cases were diagnosed as having iron deficiency with an overall frequency of 7.2 %. By using the Hardy –Weinberg equation; we found the expected number of children born with homozygous βeta-thalassaemia would be (0.3/1000 from those born) and homozygous sickle cell disease would be (0.01/1000 from those born). Conclusions: β-thalassaemia trait represented the most frequent Haemoglobinopathy in the region, Iron deficiency was significantly higher in females than in males, HPLC is a good technique for routine use and the expected number of children born with homozygous β-thalassaemia, sickle cell and others (HbD, HbE, HbH) were (0.3/1000, 0.01/1000 and 0.0085/1000 from those born) respectively.

Liver involvement in inflammatory bowel disease: What should the clinician know?

Inflammatory bowel disease (IBD) may show a wide range of extraintestinal manifestations. In this context, liver involvement is a focal point for both an adequate management of the disease and its prognosis, due to possible serious comorbidity. The association between IBD and primary sclerosing cholangitis is the most known example. This association is relevant because it implies an increased risk of both colorectal cancer and cholangiocarcinoma. Additionally, drugs such as thiopurines or biologic agents can cause drug-induced liver damage; therefore, this event should be considered when planning IBD treatment. Additionally, particular consideration should be given to the evidence that IBD patients may have concomitant chronic viral hepatitis, such as hepatitis B and hepatitis C. Chronic immunosuppressive regimens may cause a hepatitis flare or reactivation of a healthy carrier state, therefore careful monitoring of these patients is necessary. Finally, the spread of obesity has involved even IBD patients, thus increasing the risk of non-alcoholic fatty liver disease, which has already proven to be more common in IBD patients than in the non-IBD population. This phenomenon is considered an emerging issue, as it will become the leading cause of liver cirrhosis.

Transmitted Water Disease, Assessment of Immunopathogenesis of Chronic Hepatitis B and The Carrier State of Disease

The transmission of viral hepatitis type B (HBV) is of significant public health concern. The infection result depends on how well the virus interacts with the host and in particular, on the ability to respond inherently and adaptively to the humoral and cellular immunity. The purpose of this study is to evaluate clinical, immunology and tracer status (viral). This study showed the relationship between the immune and chronic conditions of Iraqi patients who are chronic hepatitis virus B or HBV carriers. The study included (111) chronically-viral hepatitis type (b) and (112) hepatitis virus surface antigen type (b) healthy carriers from out of patients. The result of this study proved that a non-significant correlation was observed between cellular immune response (CD4 and CD8) among chronic hepatitis B patients. For CD8+ lymphocytes: there was a highly significant decrease (P&lt;0.001) in the percentage means of the CD8+ cells in CHB patients, as compared with the carrier groups. For CD4+ lymphocytes: there was a slight decrease in the percentage of these lymphocytes in the peripheral blood of the patients, as compared with the carrier groups, a non-significant importance was recorded between them. The percentage of cytotoxic T-lymphocyte CD8+ was significantly decreased in CHB patients as compared to the carrier group. One of the deciding factors for the form of infection, and the immune response, which developed in accordance with a number of other biochemical factors and genes is chronic hepatitis B immunopathogénesis and carrier condition with the level of cellular immunity.

Hemoglobinopathy gone astray—three novel forms of α-thalassemia in Norwegian patients characterized by quantitative real-time PCR and DNA sequencing

α-thalassemia is one of the most common monogenic diseases worldwide and is caused by reduced or absent synthesis of α-globin chains, most commonly due to deletions of one or more of the α-globin genes. α-thalassemia occurs with high frequency in tropical and subtropical regions of the world and are very rarely found in the indigenous Scandinavian population. Here, we describe four rare forms of α-thalassemia out of which three are novel, found in together 20 patients of Norwegian origin. The study patients were diagnosed during routine hemoglobinopathy evaluation carried out at the Department of Medical Biochemistry, Oslo University Hospital, Norway. The patients were selected for their thalassemic phenotype, despite Norway as country of origin. All samples went through standard hemoglobinopathy evaluation. DNA sequencing and copy number variation (CNV) analysis using quantitative real-time polymerase chain reaction (qPCR) was applied to detect sequence variants and uncommon deletions in the α-globin gene cluster, respectively. Deletion breakpoints were characterized using gap-PCR and DNA sequencing. DNA sequencing revealed a single nucleotide deletion in exon 3 of the HBA2 gene (NM_000517.4(HBA2):c.345del) and a novel deletion of 20 nucleotides in exon 2 of the HBA2 gene (NM_000517.4(HBA2):c.142_161del). qPCR CNV analysis detected two novel large deletions in the α-globin gene cluster, –(NOR) deletion covering both α-globin genes and (αα)Aurora Borealis affecting the regulatory region, leaving the downstream α-globin genes intact. Even though inherited globin gene disorders are extremely rare in indigenous Scandinavians, the possibility of a carrier state should not be ignored.

Suppressing the Dark Current in Quantum Dot Infrared Photodetectors by Controlling Carrier Statistics

AbstractLead sulfide colloidal quantum dot photodiodes (PbS QDPDs) exhibit a high energy conversion efficiency for infrared detection. Despite the high photoinduced current, the performance of PbS QDPDs is limited by the high dark current which is rarely investigated. Understanding the dark current in PbS QDPDs is critical to improving the detectivity of PbS QDPDs. Herein, it is demonstrated that minority carriers of I‐passivated PbS films and trap sites of EDT‐passivated PbS films are related to the dark current of PbS QDPD. Utilizing annealing and low‐temperature ligand exchange processes, the dark current density can be decreased almost tenfold by suppressing the minority carrier diffusion in the PN junction and trap‐assisted charge injection from the electrode. PN junction simulation, space charge limited current measurements, as well as structural, optical, and chemical characterizations are conducted to elucidate the origins of the dark current suppression. The authors achieve the lowest dark current density of 2.9 × 10−5 mA cm−2 at −1 V among PbS‐based QDPDs and a high detectivity of 6.7 × 1012 Jones at 980 nm. It is believed that this work provides fundamental understanding of carrier statistics in nanomaterials and device performance as well as a technological basis for realizing low‐cost high‐performance optoelectronic devices.

Base Editing Repairs the HbE Mutation Restoring the Production of Normal Globin Chains in Severe HbE/β-Thalassemia Patient Hematopoietic Stem Cells and Erythroid Cells

HbE/β-thalassemia is the commonest form of severe β-thalassemia, and comprises approximately 50% of all cases worldwide. HbE/β-thalassemia is caused by the HbE codon 26 G>A mutation on one allele and any severe β 0-thalassemia mutation on the other. These mutations lead to a reduction in β-globin production, resulting in a relative excess in α-globin chains that go on to cause ineffective erythropoiesis. Importantly, individuals with a mutation on one, but not two, alleles have β-thalassemia trait, a carrier state with a normal phenotype. Recent gene therapy and gene editing approaches have been developed to treat β-thalassemia but do not directly repair the causative mutation in-situ. Gene replacement approaches rely on lentiviral vector-based sequence insertion or homology directed repair (HDR). HbF induction strategies also rely on non-homologous end joining (NHEJ) targeting of enhancers in-trans. These approaches, whilst variably successful, are associated with potential safety concerns. Adenine base editors (ABEs) potentially circumvent these problems by directly repairing pathogenic variants in-situ through deamination. ABEs catalyse A-T to G-C conversions through targeting with a Cas9-nickase and single-guide RNA (sgRNA). Conversion of the HbE codon to normal through base editing is an attractive strategy to recapitulate the phenotypically normal β-thalassemia trait state without potentially harmful double-strand breaks or random vector insertions (Figure 1A). ABEs are able to convert the HbE codon (AAG, lys) to wild-type (GAG, glu), but also to GGG (gly) or AGG (arg). GGG at codon 26 is found in a naturally occurring hemoglobin, Hb Aubenas. Heterozygotes have normal red cell indices and are phenotypically normal.We electroporated the latest generation of ABE8 editors (ABE8e, ABE8.13 and ABE8 V106W) as mRNA into WT CD34+ hematopoietic stem and progenitor cells (HSPCs) with sgRNAs targeting the middle A of the WT GAG codon. These had similar editing efficiencies although ABE8 V106W had marginally higher on-target efficiency. V106W has been evolved to have a favourable off-target profile. V106W mRNA/sgRNA was electroporated into 3 different severe HbE/β-thalassemia donor HSPCs. The HbE codon was converted to WT with a mean 28.7% efficiency, to Hb Aubenas 48.6% and to an undescribed AGG codon 2.1%. The mean conversion from HbE to a normal or normal variant was 78.7±8.7% (Figure 1B). The indel rate from inadvertent on-target Cas9 cleavage was below 0.5%. Edited cells did not show any perturbations in erythroid differentiation as assessed by Immunophenotyping and cellular morphology. In differentiated erythroid cells, RT-qPCR showed a mean fall in the α/β mRNA ratio to 0.65±0.08 (unedited patient cells normalised to 1, n=5, Figure 1C), indicating a reduction in the relative excess α-globin gene expression. Protein analysis by CE-HPLC showed a 3.6-fold reduction in HbE levels (SD±1.3) and a 13.5-fold increase in HbA/Hb Aubenas (SD±2.4, Figure 1C and D).To prove that base editing using mRNA was possible in long-term HSCs, CD34+ cells from 4 WT cord blood donors were edited using ABEmax. Mice were culled after 16 weeks, and human cells were collected and transplanted into 7 secondary mice, which were also culled after 16 weeks. Each secondary mouse showed the presence of hCD45+ cells, indicating engraftment of LT-HSCs. All secondary replicates showed editing, with a mean editing efficiency of 34.5% (initial editing 46.3%). In both rounds of mice, there was robust lymphoid and myeloid engraftment and expected levels of erythroid engraftment for the NSG model in bone marrow and spleen. Potential off-target effects were assessed in-vitro by CIRCLE-seq in triplicate. These sites were assessed by targeted oligonucleotide capture of DNA from mRNA edited patient cells to detect in-vivo editing.Together these data provide robust evidence for base editing as an effective and safe therapeutic strategy for HbE/β-thalassemia. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Hypoxia Impact on Red Blood Cell-Mediated Microvascular Occlusion and Adhesion in Sickle Cell Disease and Sickle Cell Trait

Introduction: Sickle cell disease (SCD) is a recessively inherited anemia caused by a single gene mutation leading to sickle hemoglobin production. Sickle cell trait (SCT) is the carrier state. Abnormal hemoglobin polymerization and resultant red blood cell (RBC) sickling, decreased deformability and increased adhesion, are well-known features of homozygous SCD. However, the overall pathophysiological impact of SCT on the RBC remains incompletely characterized. Here we use microfluidic techniques designed by us, the OcclusionChip and SCD Biochip (previously published), and commercially available ektacytometry to investigate hypoxia impact on RBC biophysical properties in SCT.Methods: Venous blood samples were collected in EDTA from subjects with homozygous HbSS, SCT (HbAS), and non-anemic controls (HbAA) under an IRB-approved protocol. OcclusionChip devices were fabricated using standard soft lithography protocols [1]. RBCs were isolated from whole blood, re-suspended in PBS at 20% hematocrit, and passed through the OcclusionChip device with a constant inlet pressure. Following a wash step, the OcclusionChip microchannel was imaged, and Occlusion Index (OI), a standardized generalizable parameter we developed, representing the overall microcapillary network occlusion, was quantified. SCD Biochip microchannels were fabricated by lamination and were functionalized with human laminin (LN-511) [2]. Undiluted whole blood was injected into the microchannel at 1 dyne/cm 2, a shear stress value typically observed in the post-capillary venules. Following a wash step, the SCD Biochip microchannel was imaged, and the number of adherent RBCs in a 32-mm 2 window was quantified. For hypoxia experiments, a hypoxic setup was fabricated for blood deoxygenation (pO 2 ~45 mmHg) [3, 4]. Ektacytometry measurements were performed according to the manufacturers' specifications (Lorrca Maxsis). Data are reported as mean ± standard deviation (SD).Results: We initially analyzed RBC-mediated microvascular occlusion under normoxia or hypoxia using the OcclusionChip (Figure 1A). Under normoxia, HbSS-containing RBCs had relatively greater OI values compared to HbAA- and HbAS-containing RBCs (Figure 1B, P = 0.057 for HbSS vs HbAA and P = 0.060 for HbSS vs HbAS). However, exposure to hypoxia led to significantly elevated OI values in the HbAS- and HbSS-containing RBCs (Figure 1B, 0.05 ± 0.02% vs 33.62 ± 18.31%, P = 0.015 for HbAS, and 0.27 ± 0.24% vs 49.37 ± 24.47%, P = 0.001 for HbSS, normoxia vs hypoxia). Negligible occlusion was observed in HbAA-containing RBCs (Figure 1B). We then analyzed RBC adhesion to LN under normoxia or hypoxia using the SCD Biochip (Figure 1C). Hypoxia led to greater number of adherent RBCs on LN in the HbSS-containing RBCs (Figure 1D, 141 ± 91 vs 497 ± 392, P = 0.089, normoxia vs hypoxia), but this effect was not present in HbAA- or HbAS-containing RBCs (Figure 1B, 2 ± 1 vs 3 ± 1, P > 0.05 for HbAA, and 10 ± 7 vs 12 ± 3, P > 0.05 for HbAS, normoxia vs hypoxia). Further, under normoxia, we found that the HbAS-containing RBCs had slightly greater number of adherent RBCs on LN compared to the HbAA-containing RBCs (Figure 1D, P = 0.057 for HbAA vs HbAS). As previously reported, HbSS-containing RBCs showed greatest adhesion to LN under normoxia compared to the HbAA- and HbAS-containing RBCs (Figure 1D, P = 0.027 for HbSS vs HbAA and P = 0.033 for HbSS vs HbAS)., Finally, we preformed Lorrca oxyscan and found that ektacytometry is less sensitive to RBC deformability change under hypoxia in SCT (Figure 1E).Conclusions: Findings in this study suggest that although RBCs from subjects with SCT are deformable under normoxia and are able to clear narrow capillaries similar to normal RBCs, hypoxia changes deformability, presumably due to hypoxic polymer formation, and could contribute to microvascular occlusion in SCT. The OcclusionChip is a single cell-based technology, and may be more sensitive to single RBC deformability. Future studies will prospectively focus on analyzing RBC adhesion on activated microvascular endothelial cells in physiologic flow to further interrogate the impact of hypoxia on pathophysiology in SCT.

Covid Reinfection versus Asymptomatic Carrier State: A Case Report

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), first discovered in China in December 2019, has been implicated in the current coronavirus disease 2019 (COVID-19) pandemic. Although much has been learned about the virus which peaked with the development of the vaccine, there is still a lot of unanswered questions. Maximum duration of positive SARS-CoV-2 real-time reverse transcriptase polymerase chain reaction (RT-PCR) from symptom onset may be up to 3 months [1], however it is not known if the continued detection of the viral genome implies prolonged infectivity or presence of a non-viable virus [2]. Most people with COVID-19 develop antibodies after resolution of acute infection [2]. The exact duration of these antibodies in the body is unknown, but some studies have shown that both memory T-cells and B-cells can persist up to 6 to 8 months after acute SARS-CoV-2 infection [3]. These SARS-CoV-2 antibodies may confer some immunity to the person after the acute infection and have been associated with protection against subsequent infection in nonhuman primates by the same viral strain during the early recovery phase [4]. In humans, however, it is unknown to what extent this immune response indicates a protective immunity to subsequent infection with SARS-CoV-2 [5]. Few cases of reinfection have been documented worldwide with varying symptom severity; the first case in the US was published in January 2021 (reinfection occurred in June 2020) [5]. None of the initial cases reported the presence of SARS-CoV-2 antibodies at the time of reinfection. We present a patient who tested positive to SARS-CoV-2 RT-PCR twice in 10 months (Table 1). At both times, she was asymptomatic and the second time, she had coexisting SARS-CoV-2 antibodies.

Revisiting Persistent Salmonella Infection and the Carrier State: What Do We Know?

One characteristic of the few Salmonella enterica serovars that produce typhoid-like infections is that disease-free persistent infection can occur for months or years in a small number of individuals post-convalescence. The bacteria continue to be shed intermittently which is a key component of the epidemiology of these infections. Persistent chronic infection occurs despite high levels of circulating specific IgG. We have reviewed the information on the basis for persistence in S. Typhi, S. Dublin, S. Gallinarum, S. Pullorum, S. Abortusovis and also S. Typhimurium in mice as a model of persistence. Persistence appears to occur in macrophages in the spleen and liver with shedding either from the gall bladder and gut or the reproductive tract. The involvement of host genetic background in defining persistence is clear from studies with the mouse but less so with human and poultry infections. There is increasing evidence that the organisms (i) modulate the host response away from the typical Th1-type response normally associated with immune clearance of an acute infection to Th2-type or an anti-inflammatory response, and that (ii) the bacteria modulate transformation of macrophage from M1 to M2 type. The bacterial factors involved in this are not yet fully understood. There are early indications that it might be possible to remodulate the response back towards a Th1 response by using cytokine therapy.

Is There a Relation between 677C>T Polymorphism in the MTHFR Gene and the Susceptibility to Epilepsy in Young Patients? A Meta-Analysis.

Background: Numerous data show a role for genetic polymorphisms in the development of epilepsy. Previously, the TT genotype of the MTHFR 677C>T polymorphism was found to be associated with a decreased leucocyte DNA methylation status. Polymorphisms in the MTHFR gene could modify the pharmacodynamics of many drugs. This meta-analysis aimed to assess the relationship between MTHFR 677C>T polymorphism and susceptibility to epilepsy in young patients. Methods: Available databases (PubMed, Embase, Google Scholar, SciELO, and Medline) were searched using specific keywords. Eight studies, published between 1999 and 2019, with 1678 young patients with epilepsy and 1784 controls, met the inclusion criteria. Apart from the total groups, additional analyses in age subgroups (i.e., young adults and children) were conducted. Statistical analyses were conducted using the RevMan 5.4 and MedCalc software. The pooled odds ratio (OR) was estimated with a random- or fixed-effects model depending on the heterogeneity. Analyses were performed for five genetic models, i.e., dominant (CT + TT vs. CC), recessive (TT vs. CC + CT), additive (TT vs. CC), heterozygous (CT vs. CC), and allelic (T vs. C). The publication bias was assessed with the use of Egger’s and Begg’s tests. Results: Both the MTHFR TT genotype (in the additive model) and the T allele (in the allelic model) significantly increased the risk of epilepsy when the total groups were compared (OR = 1.44, p = 0.002, and OR = 1.183, p = 0.001, respectively). The sensitivity analysis for these models indicated the stability of the results. Similarly, significant results were obtained among young adults for all the genetic models (dominant model: OR = 1.28, p = 0.002; recessive model: OR = 1.48, p = 0.003; additive model: OR = 1.63, p < 0.001; heterozygous model: OR = 1.21, p = 0.028; and allelic model: OR = 1.256, p < 0.001). Those results were also stable and reliable. In the group of children, no relation between 677C>T polymorphism and epilepsy was observed; however, the analysis was based only on three studies, and one study also comprised young adults. No publication bias was demonstrated. Conclusions: The meta-analysis revealed that the carrier state for the T allele as well as the TT genotype of the MTHFR 677C>T polymorphism increases the risk of epilepsy in young adults but not in children.