Red Cell Protein Research Articles

Label-free photoacoustic microscopy: a potential tool for the live imaging of blood disorders in zebrafish

The zebrafish has emerged as a useful model for human hematological disorders. Transgenic zebrafish that express green fluorescence protein (GFP) in red blood cells (RBCs) visualized by fluorescence microscopy (FLM) is a fundamental approach in such studies to understand the cellular processes and biological functions. However, additional and cumbersome efforts are required to breed a transgenic zebrafish line with reliable GFP expression. Further, the yolk autofluorescence and finite GFP fluorescence lifetimes also have an adverse impact on the observation of target signals. Here, we investigate the identification of intracerebral hemorrhage (ICH) and hemolytic anemia (HA) in zebrafish embryos using label-free photoacoustic microscopy (PAM) for imaging. First, ICH and HA in transgenic LCR-EGFP zebrafish are mainly studied by PAM and FLM. The results show that PAM is comparable to FLM in good identification of ICH and HA. Besides, PAM is more advantageous in circumventing the issue of autofluorescence. Secondly, ICH and HA in the transparent casper zebrafish without fluorescent labeling are imaged by PAM and bright-field microscopy (BFM). Because of the high contrast to reveal RBCs, PAM obviously outperforms BFM in the identification of both ICH and HA. Note that FLM cannot observe casper zebrafish due to its lack of fluorescent labeling. Our work proves that PAM can be a useful tool to study blood disorders in zebrafish, which has advantages: (i) Reliable results enabled by intrinsic absorption of RBCs; (ii) wide applicability to zebrafish strains (no requirement of a transgene); (iii) high sensitivity in identification of ICH and HA compared with BFM.

The interactome of the N-terminus of band 3 regulates red blood cell metabolism and storage quality.

Band 3 (anion exchanger 1; AE1) is the most abundant membrane protein in red blood cells, which in turn are the most abundant cells in the human body. A compelling model posits that, at high oxygen saturation, the N-terminal cytosolic domain of AE1 binds to and inhibits glycolytic enzymes, thus diverting metabolic fluxes to the pentose phosphate pathway to generate reducing equivalents. Dysfunction of this mechanism occurs during red blood cell aging or storage under blood bank conditions, suggesting a role for AE1 in the regulation of the quality of stored blood and efficacy of transfusion, a life-saving intervention for millions of recipients worldwide. Here we leveraged two murine models carrying genetic ablations of AE1 to provide mechanistic evidence of the role of this protein in the regulation of erythrocyte metabolism and storage quality. Metabolic observations in mice recapitulated those in a human subject lacking expression of AE11-11 (band 3 Neapolis), while common polymorphisms in the region coding for AE11-56 correlate with increased susceptibility to osmotic hemolysis in healthy blood donors. Through thermal proteome profiling and crosslinking proteomics, we provide a map of the red blood cell interactome, with a focus on AE11-56 and validate recombinant AE1 interactions with glyceraldehyde 3-phosphate dehydrogenase. As a proof-of-principle and to provide further mechanistic evidence of the role of AE1 in the regulation of redox homeo stasis of stored red blood cells, we show that incubation with a cell-penetrating AE11-56 peptide can rescue the metabolic defect in glutathione recycling and boost post-transfusion recovery of stored red blood cells from healthy human donors and genetically ablated mice.

Borate transporters as models for studying SLC4 transport mechanisms

Borate transport proteins regulate the availability of borate, an essential plant micronutrient that is toxic in excess. Borate transporters share homology with SLC4 transporters, the archetypal member of which is human Band 3, or Anion Exchanger 1 (AE1). AE1 is the most abundant membrane protein in red blood cells where it exchanges bicarbonate and chloride, and mutations in AE1 are linked to several diseases. In this study we tested whether the S. cerevisiae Bor1 transporter (ScBor1) shares key features in common with the human AE1 transporter (HsAE1) to determine the extent to which ScBor1 serves as a relevant experimental model. Here we show evidence that, like HsAE1, ScBor1 is inhibited by the stilbene derivatives SITS and DIDS. Additionally, we demonstrate that the ScBor1 D347 residue, which is homologous to the essential E681 of HsAE1, can be complemented by the D347E but not D347N substitution. Lastly, we identify several new amino acids critical for function. These data collectively highlight similarities in the biochemical features of ScBor1 and HsAE1 and help elucidate their mechanisms.

A Dynamic Bayesian Model for Identifying High-Mortality Risk in Hospitalized COVID-19 Patients.

As Coronavirus Disease 2019 (COVID-19) hospitalization rates remain high, there is an urgent need to identify prognostic factors to improve patient outcomes. Existing prognostic models mostly consider the impact of biomarkers at presentation on the risk of a single patient outcome at a single follow up time. We collected data for 553 Polymerase Chain Reaction (PCR)-positive COVID-19 patients admitted to hospital whose eventual outcomes were known. The data collected for the patients included demographics, comorbidities and laboratory values taken at admission and throughout the course of hospitalization. We trained multivariate Markov prognostic models to identify high-risk patients at admission along with a dynamic measure of risk incorporating time-dependent changes in patients’ laboratory values. From the set of factors available upon admission, the Markov model determined that age >80 years, history of coronary artery disease and chronic obstructive pulmonary disease increased mortality risk. The lab values upon admission most associated with mortality included neutrophil percentage, red blood cells (RBC), red cell distribution width (RDW), protein levels, platelets count, albumin levels and mean corpuscular hemoglobin concentration (MCHC). Incorporating dynamic changes in lab values throughout hospitalization lead to dramatic gains in the predictive accuracy of the model and indicated a catalogue of variables for determining high-risk patients including eosinophil percentage, white blood cells (WBC), platelets, pCO2, RDW, large unstained cells (LUC) count, alkaline phosphatase and albumin. Our prognostic model highlights the nuance of determining risk for COVID-19 patients and indicates that, rather than a single variable, a range of factors (at different points in hospitalization) are needed for effective risk stratification.

Tyrosine Phosphorylation Modulates Peroxiredoxin-2 Activity in Normal and Diseased Red Cells.

Peroxiredoxin-2 (Prx2) is the third most abundant cytoplasmic protein in red blood cells. Prx2 belongs to a well-known family of antioxidants, the peroxiredoxins (Prxs), that are widely expressed in mammalian cells. Prx2 is a typical, homodimeric, 2-Cys Prx that uses two cysteine residues to accomplish the task of detoxifying a vast range of organic peroxides, H2O2, and peroxynitrite. Although progress has been made on functional characterization of Prx2, much still remains to be investigated on Prx2 post-translational changes. Here, we first show that Prx2 is Tyrosine (Tyr) phosphorylated by Syk in red cells exposed to oxidation induced by diamide. We identified Tyr-193 in both recombinant Prx2 and native Prx2 from red cells as a specific target of Syk. Bioinformatic analysis suggests that phosphorylation of Tyr-193 allows Prx2 conformational change that is more favorable for its peroxidase activity. Indeed, Syk-induced Tyr phosphorylation of Prx2 enhances in vitro Prx2 activity, but also contributes to Prx2 translocation to the membrane of red cells exposed to diamide. The biologic importance of Tyr-193 phospho-Prx2 is further supported by data on red cells from a mouse model of humanized sickle cell disease (SCD). SCD is globally distributed, hereditary red cell disorder, characterized by severe red cell oxidation due to the pathologic sickle hemoglobin. SCD red cells show Tyr-phosphorylated Prx2 bound to the membrane and increased Prx2 activity when compared to healthy erythrocytes. Collectively, our data highlight the novel link between redox related signaling and Prx2 function in normal and diseased red cells.

A Review on Epidemiology and Thalassemia Distribution, in Relevance to United Arab Emirates Population

Thalassemia is a blood disorder passed down through families (inherited) in which the body makes an abnormal form or inadequate amount of hemoglobin. Hemoglobin is the protein in red blood cells that carries oxygen. The disorder results in large numbers of red blood cells being destroyed, which leads to anemia. Depending on the type of mutated globin, there are two types of thalassemia: Alpha- (α-) thalassemia and beta- (β-) thalassemia. It is estimated that around 9 million people suffering from thalassemia become pregnant every year worldwide, of which 1.33 million are at risk of developing severe thalassemia disorder. Thalassemia is a problem in the health sector in many Middle East and North Africa (MENA), region Southeast Asia, Central Asia and the Indian subcontinent. Countries in the MENA region, particularly the Gulf Cooperation Council (GCC) countries and the UAE, have higher prevalence of β-thalassemia and carriers than α-thalassemia. The UAE has the most prevalence of thalassemia carrier among all the MENA countries, due to a huge rate of consanguineous marriage. The UAE government has initiated various measure to reduce the incidences of thalassemia among which one such program included launching of a national campaign in 2008 to promote premarital screening that provides non-instructive genetic counseling to non-at-risk couples. Despite various measures of the thalassemia premarital screening program, the premarital screening program has not been able to effectively reduce the frequency of marriages in high-risk couples in UAE. The study concludes that targeting young people and raising awareness of thalassemia and increasing its burden may reduce inter-carrier marriage and reduce the incidence of such disorders.

An investigation of traditional uses and anti-inflammatory property of Clematis buchananiana De Candolle and Tupistra nutans Wall. ex Lindl.: Native ethnomedicinal plants from Sikkim, India

In the traditional medicinal system of Sikkim Clematis buchananiana and Tupistra nutans is used extensively to treat various ailments, however, they have not been validated for their anti-inflammatory property by in vitro method. Therefore, the present study was carried out to investigate specific ethnomedicinal uses, in-vitro anti-inflammatory property, and phytochemical constituents of Clematis buchananiana and Tupistra nutans . The ethnomedicinal usage was studied by calculating the value for fidelity level, use-value, and informant consensus factor. Stabilization of human red blood cell membrane and protein denaturation method was used to study anti-inflammatory property. The phytochemicals were analysed by the methods described elsewhere. Clematis buchananiana was found to be used more frequently for sinusitis, headache, cold and Tupistra nutans for high blood pressure, diabetes and stomach-ache. Both C. buchananiana and T. nutans was found to inhibit the HRBC membrane and protein denaturation effectively in a dose-dependent manner. However, inhibition of haemolysis and protein denaturation by C. buchananiana was found to be higher than T. nutans at all doses. The phytochemical screening revealed the presence of anti-inflammatory metabolites such as flavonoids and phenolics in both the plants. The results provide evidence for the anti-inflammatory property of C. buchananiana and T. nutans .

An investigation of traditional uses and anti-inflammatory property of Clematis buchananiana De Candolle and Tupistra nutans Wall. ex Lindl.: Native ethnomedicinal plants from Sikkim, India

In the traditional medicinal system of Sikkim Clematis buchananiana and Tupistra nutans is used extensively to treat various ailments, however, they have not been validated for their anti-inflammatory property by in vitro method. Therefore, the present study was carried out to investigate specific ethnomedicinal uses, in-vitro anti-inflammatory property, and phytochemical constituents of Clematis buchananiana and Tupistra nutans . The ethnomedicinal usage was studied by calculating the value for fidelity level, use-value, and informant consensus factor. Stabilization of human red blood cell membrane and protein denaturation method was used to study anti-inflammatory property. The phytochemicals were analysed by the methods described elsewhere. Clematis buchananiana was found to be used more frequently for sinusitis, headache, cold and Tupistra nutans for high blood pressure, diabetes and stomach-ache. Both C. buchananiana and T. nutans was found to inhibit the HRBC membrane and protein denaturation effectively in a dose-dependent manner. However, inhibition of haemolysis and protein denaturation by C. buchananiana was found to be higher than T. nutans at all doses. The phytochemical screening revealed the presence of anti-inflammatory metabolites such as flavonoids and phenolics in both the plants. The results provide evidence for the anti-inflammatory property of C. buchananiana and T. nutans .

A novel platform for peptide-mediated affinity capture and LC-MS/MS identification of host receptors involved in Plasmodium invasion

Successful Plasmodium falciparum invasion of red blood cells includes the orderly execution of highly specific receptor-ligand molecular interactions between the parasite's proteins and the red blood cell membrane proteins. There is a growing need for elucidating receptor-ligand pairings, which will help in understanding the parasite's biology and provide the fundamental basis for developing prophylactic or therapeutic alternatives leading to mitigating or eliminating this type of malaria.We have thus used Plasmodium falciparum RH5 - derived peptides and ghost red blood cell proteins in synthetic peptide affinity capture assays to identify important host receptors used by Plasmodium spp. in the invasion of red blood cells. LC-MS/MS analysis confirmed the extensively described interaction between PfRH5 and the basigin receptor on the red blood cell membrane.As shown here, tagged synthetic peptides displaying high binding ability to erythrocytes can be used to identify receptors present in protein extracts from ghost red blood cells via affinity capture and LC-MS/MS. SignificanceThe article describes a novel approach for identifying red blood cell receptors based on the ability of synthetic peptides having high red blood cell binding capacity to capture Plasmodium spp. receptors on proteins extracted from ghost red blood cells. Specifically, novel methods to identify Plasmodium falciparum reticulocyte binding protein homolog 5 PfRH5 and basigin interaction using a combination of affinity capture and LC-MS/MS assays is described. Identification of these host RBC receptors interacting with malarial parasite proteins is of utmost importance in studying the disease's pathogenesis and will provide crucial information in understanding the parasite's biology. In addition, data from these studies can be used to identify potential therapeutic target(s) to mitigate or eliminate this debilitating disease.

Effects of exercise on hematological and serum biochemical parameters in riding ponies

The purpose of the present study was to investigate changes in hematological and serum biochemical parameters in ponies exercising 3 times a day. The study included 10 healthy 4- to 5-year-old Jeju crossbred ponies used in riding lessons at an equestrian riding school. Hematology and serum chemistry samples were obtained before the initial lesson and after the last lesson of the day. The post-exercise results showed that packed cell volume, white blood cell, red blood cell, hemoglobin, and total protein levels increased significantly (p < 0.05). Serum Na+ also increased significantly (p < 0.01), but serum Ca++ decreased significantly (p < 0.01). Creatinine kinase, aspartate amino transferase, gamma glutamyl transferase, blood urea nitrogen, creatinine, and bilirubin levels increased significantly (p < 0.05), but the glucose level decreased significantly (p < 0.05). Furthermore, the serum cortisol hormone level increased significantly (p < 0.01). The results suggest that participating in riding lessons three times a day may result in various physiological changes, indicating the presence of exercise-related stress in riding ponies. Keywords: riding horses, exercising stress, hematological, serum biochemical, cortisol

GC-MS and LC-MS/MS pilot studies on the guanidine (NG)-dimethylation in native, asymmetrically and symmetrically NG-dimethylated arginine-vasopressin peptides and proteins in human red blood cells

Protein-arginine methyltransferases catalyze the methylation of the guanidine (NG) group of proteinic L-arginine (Arg) to produce monomethyl and dimethylarginine proteins. Their proteolysis releases the free amino acids monomethylarginine (MMA), symmetric dimethylarginine (SDMA) and asymmetric dimethylarginine (ADMA), respectively. MMA, SDMA and ADMA are inhibitors of the nitric oxide synthase (NOS) activity. High circulating and low urinary concentrations of ADMA and SDMA are considered risk factors in the cardiovascular and renal systems, mainly due to their inhibitory action on NOS activity. Identity, biological activity and concentration of NG-methylated proteins are largely unknown. The present study addressed these issues by using GC-MS and LC-MS/MS approaches. GC-MS was used to quantify free ADMA released by classical HCl-catalyzed hydrolysis of three synthetic Arg-vasopressin (V) peptides and of unknown endogenous NG-dimethylated proteins. The cyclic (c) disulfide forms of Arg-vasopressin analogs, i.e., Arg-vasopressin (cV-Arg-Gly-NH2), asymmetrically NG-dimethylated vasopressin (cV-ADMA-Gly-NH2) and symmetrically NG-dimethylated vasopressin (cV-SDMA-Gly-NH2) were used as model peptides in quantitative GC-MS analyses of ADMA, SDMA and other expected amino acids from the hydrolyzed Arg-vasopressin analogs. cV-ADMA-Gly-NH2 and cV-SDMA-Gly-NH2 were discriminated from cV-Arg-Gly-NH2 by LC-MS and LC-MS/MS, yet they were indistinguishable from each other. The same applies to the respective open (o) reduced and di-S-acetamide forms of oV-ADMA-Gly-NH2, oV-SDMA-Gly-NH2 and oV-Arg-Gly-NH2. Our LC-MS and LC-MS/MS studies suggest that the Arg-vasopressin analogs form [(M−H)]+ and [(M−H)+H]+ in the positive ESI mode and undergo in part conversion of their terminal Gly-NH2 (NH2, 16 Da) group to Gly-OH (OH, 17 Da). The product ion mass spectra of the di-S-acetamide forms are complex and contain several intense mass fragments differing by 1 Da. cV-ADMA-Gly-NH2 and cV-SDMA-Gly-NH2 induced platelet aggregation in platelet-rich human plasma with moderately different initial velocity and maximal aggregation rates compared to cV-Arg-Gly-NH2. Previous studies showed that human red blood cells are rich in large (>50 kDa) ADMA-containing proteins of unknown identity. Our LC-MS/MS proteomic study identified several membrane and cytosolic erythrocytic NG-dimethylated proteins, including spectrin-α (280 kDa), spectrin-β (247 kDa) and protein 4.1 (80 kDa). Being responsible for the stability of the erythrocyte membrane, the newly identified main targets for NG-dimethylation in human erythrocytes should be given a closer look in erythrocytic diseases like hereditary spherocytosis.

Optimizing red blood cell protein extraction for biomarker quantitation with mass spectrometry.

Red blood cells (RBC) are the most common cell type found in blood. They might serve as reservoir for biomarker research as they are anuclear and lack the ability to synthesize proteins. Not many biomarker assays, however, have been conducted on RBC because of their large dynamic range of proteins, high abundance of lipids, and hemoglobin interferences. Here, we developed a semiquantitative mass spectrometry-based assay that targeted 144 proteins and compared the efficiency of urea, sodium deoxycholate, acetonitrile, and HemoVoid™ in their extraction of the RBC proteome. Our results indicate that protein extraction with HemoVoid™ led to hemoglobin reduction and increased detection of low abundance proteins. Although hemoglobin interference after deoxycholate and urea extraction was high, there were adequate amounts of low abundance proteins for quantitation. Extraction with acetonitrile led to an overall decrease in protein abundances probably as a result of precipitation. Overall, the best compromise in sensitivity and sample processing time was achieved with the urea-trypsin digestion protocol. This provided the basis for large-scale evaluations of protein targets as potential blood-based biomarkers. As a proof of concept, we applied this assay to determine that alpha-synuclein, a prominent marker in Parkinson's disease, has an average concentration of approximately 40μgmL-1 in RBC. This is important to know as the concentration of alpha-synuclein in plasma, typically in the picogram per milliliter range, might be partially derived from lysed RBC. Utilization of this assay will prove useful for future biomarker studies and provide a more complete analytical toolbox for the measurement of blood-derived proteins. Graphical abstract.

Сравнительная характеристика липидного состава и морфофункциональных показателей ядерных и безъядерных эритроцитов в условиях гипоксии

A comparative study of the properties of nuclear and non-nuclear erythrocytes in experimental hypoxia. The phospholipid and fatty acid composition of individual phospholipids, the content of free fatty acids in the composition of nuclear-free and nuclear red blood cells were determined by thin-layer chromatography. The method of laser interference microscopy was used to study the morphometric characteristics of nuclear and nuclear-free red blood cells and the distribution of hemoglobin. Conformational changes in the hemoporphyrin of nuclear and nuclear-free red blood cells under hypoxia were studied by Raman spectroscopy. It has been shown that under conditions of hypoxia, changes in the phospholipid and fatty acid composition occur in the membranes of both nuclear and non-nuclear erythrocytes, leading to the accumulation of lysoforms of phospholipids, diacylglycerol and free fatty acids, as well as to an increase in the degree of saturation of fatty acids in phospholipids. These changes indicate structural changes in the membrane of nuclear-free and nuclear red blood cells and, as a result of this, functional changes both on the part of the red blood cell in general and the main oxygen-transporting protein of red blood cells - hemoglobin.

A disposable microfluidic-integrated hand-held plasmonic platform for protein detection

Healthcare is in the midst of a transformative shift from centralized care to point-of-care (POC). In this regard, recent efforts have focused on integration of biosensing technologies with clinical management and existing healthcare systems to improve the effectiveness and quality of care. Plasmonic technologies in particular, have been used for multiple applications in biosensing, pharmaceutical industry, food quality monitoring, and healthcare. However, bulky-sized platforms, expensive instrumentation, incomprehensive benchmarking, laborious protocols, and time-consuming processing steps remain challenges to adopt biosensing platforms to the POC settings. Here, we present a hand-held biosensing platform that integrates a plasmonic detection modality with a microfluidic chip. As a biological target model, we assess hemoglobin—an iron carrying protein in red blood cells. We comprehensively perform theoretical simulations and kinetic calculations to benchmark the platform performance. Overall, this miniaturized platform provides label-free detection, simple configuration for user-interface, facile sampling, assay-time down to 15–30 min, and inexpensive disposable chips. Therefore, this platform will potentially accelerate the deployment of portable biosensing systems for the POC and primary care settings.

Changes of B cell-activating factor and a proliferation-inducing ligand in serum of children with Hen(o)ch-Sch(o)nlein purpura nephritis

Objective To investigate the changes of B cell-activating factor (BAFF) and a proliferation- inducing ligand (APRIL) in serum of children with Henoch- Schonlein purpura nephritis (HSPN), and to explore their role in the pathogenesis of children HSPN. Methods A total of 28 children with HSPN who were before treatment were selected in Department of Pediatrics Nephrology and Rheumatology, Shengjing Hospital of China Medical University from November 2017 to August 2018.Sixteen children with Henoch-Schonlein purpura were selected as HSP group, and 20 healthy children were selected as healthy control group.Followed the HSPN guideline to cure the patients for 6-8 weeks.The clinical data were collected.Serum levels of BAFF and APRIL were measured by adopting enzyme-linked immunosorbent assay (ELISA). Results (1)Changes of serum BAFF level: the serum levels of BAFF in HSPN children were significantly lower than those in the HSP group and the healthy control group[ HSPN group (0.652±0.360) μg/L, HSP group (1.276±0.459) μg/L, healthy control group (1.285±0.299) μg/L, F=17.519, P=0.000]. Moreover, the serum levels of BAFF in before treatment were significantly lower than those in after treatment [before treatment (0.652±0.360) μg/L, after treatment (0.860±0.262) μg/L, P 0.05). (2)Changes of serum APRIL level: the serum levels of APRIL in HSPN and HSP children were both significantly higher than those in healthy control group, but there were no marked differences between the 2 groups [HSPN group (2.285±1.015) μg/L, HSP group (2.609±1.264) μg/L, healthy control group (1.677±0.118) μg/L, F=3.647, P=0.016]. There were no significant differences in the serum levels of APRIL between before treatment and after treatment [ before treatment (2.285±1.015) μg/L, after treatment (2.042±0.695) μg/L, P>0.05]. (3)Pearson correlation analysis results showed that the serum levels of BAFF were negatively correlated with 24 h urinary protein, urinary microalbumin, and urine red blood cell count (r=-0.587, -0.608, -0.515, all P<0.05). The serum levels of APRIL were positively correlated with serum IgA(r=0.588, P<0.05). Conclusions The level of serum BAFF decreased and APRIL increased in children with HSPN, which was related to the degree of renal involvement.It suggests that BAFF and APRIL may be related to the pathogenesis of HSPN in children. Key words: Henoch- Schonlein purpura nephritis; Child; B cell-activating factor; Proliferation-inducing ligand

Effects of water temperature and diet on blood parameters and stress levels in hybrid grouper (Epinephelus fuscoguttatus ♀ × E. lanceolatus ♂) juveniles

Hybrid grouper (Epinephelus fuscoguttatus [TG] × E. lanceolatus [GG]) juveniles were evaluated in terms of their blood hematology and biochemical levels after 60 days of exposure to different temperatures (22 °C, 26 °C, 30 °C, and 34 °C) in triplicate (20 fish/tank; body weight 200 ± 15 g; length 22.5 ± 1 cm). The fish were fed daily with commercial pellets (42% protein, 9% ash, and 11% lipid) or shrimp (42% protein, 9% ash, and 11% lipid). Results showed that the blood hematologic parameters of the fish fed with shrimp at 26 °C were significantly better than those at other temperatures. The lowest stress level measured in terms of glucose (26 ± 10 mg dL−1) and cortisol concentrations (0.5 ± 0.1 ng mL−1) after 60 days of the experiment were observed in the fish fed with shrimp at 26 °C compared with those fed with other diets and at other temperatures. Overall, the TGGG hybrid showed the highest red blood cell and total protein counts in fish fed with a shrimp diet at 26 °C, which were healthful and less stressful conditions. Thus, the health of TGGG hybrid fed with shrimp was better than that of fish fed with other diets and at other temperatures. These findings would be useful for further studies on the management of the TGGG hybrid to increase production.

Splenectomy for people with thalassaemia major or intermedia.

Thalassaemia is a genetic disorder of the haemoglobin protein in red blood cells. It has been historically classified into thalassaemia minor, intermedia and major, depending on the genetic defect and severity of the disease. The clinical presentation of β-thalassaemia varies widely from a mild asymptomatic form in thalassaemia minor, to a severe disease in thalassaemia major where individuals are dependant on life-long blood transfusions. The hallmark of thalassaemia syndromes is the production of defective red blood cells that are removed by the spleen resulting in an enlarged hyperfunctioning spleen (splenomegaly). Removal of the spleen may thus prolong red blood cell survival by reducing the amount of red blood cells removed from circulation and may ultimately result in the reduced need for blood transfusions. To assess the efficacy and safety of splenectomy in people with β-thalassaemia major or intermedia. We searched the Cochrane Cystic Fibrosis and Genetic Disorders Review Group's Haemoglobinopathies Trials Register, compiled from searches of electronic databases and the handsearching of journals and conference abstract books. We also searched online trial registries and the reference lists of relevant articles and reviews (27 July 2018).Date of the most recent search of the Group's trials register: 02 August 2019. We included randomised controlled and quasi-randomised controlled studies of people of any age with thalassaemia major or intermedia, evaluating splenectomy in comparison to conservative treatment (transfusion therapy and iron chelation) or other forms of splenectomy compared to each other (laparoscopic, open, radio-frequency). Two authors independently selected and extracted data from the single included study using a customised data extraction form and assessed the risk of bias. The quality of the evidence was assessed using GRADE. One study, including 28 participants was included in the review; the results were described, primarily, in a narrative manner. This study assessed the feasibility of splenectomy using a laparoscopic approach versus open surgery. Given the lack of detail regarding the study methods beyond randomisation, the overall risk of bias for this study was unclear. The study was carried out over a period of 3.5 years, with each participant followed up only until discharge (less than one week after the intervention); it did not assess the majority of the outcomes outlined in this review (including two of the three primary outcomes, frequency of transfusion and quality of life). A total of three serious post-operative adverse events (the review's third primary outcome) were reported in the laparoscopic splenectomy group (one case of atelectasis and two cases of bleeding), compared to two events of atelectasis in the open surgery group; however, there were no significant differences between the groups for either atelectasis, risk ratio (RR) 0.50 (95% confidence interval (CI) 0.05 to 4.90) or for bleeding, RR 5.00 (95% CI 0.26 to 95.61) (very low-quality evidence). In addition, the study also reported three serious cases of intra-operative bleeding in the laparoscopic group which mandated conversion to open surgery, although the difference between groups was not statistically significant, RR 7.00 (95% CI 0.39 to 124.14) (very low-quality evidence). These effect estimates are based on very small numbers and hence are unreliable and imprecise. From this small study, there appeared to be an advantage for the laparoscopic approach, in terms of post-operative hospital stay, although the group difference was not large (median difference of 1.5 days, P = 0.03). The review was unable to find good quality evidence, in the form of randomised controlled studies, regarding the efficacy of splenectomy for treating thalassaemia major or intermedia. The single included study provided little information about the efficacy of splenectomy, and compared open surgery and laparoscopic methods. Further studies need to evaluate the long-term effectiveness of splenectomy and the comparative advantages of surgical methods. Due to a lack of high quality evidence from randomised controlled studies, well-conducted observational studies may be used to answer this question.

Ketogenic Diet and microRNAs Linked to Antioxidant Biochemical Homeostasis.

Recently, we demonstrated the capability of the ketogenic diet (KD) to influence the microRNA (miR) expression profile. Here, we report that KD is able to normalize miR expression in obese subjects when compared with lean subjects. By applying two different bioinformatics tools, we found that, amongst the miRs returning to comparable levels in lean subjects, four of them are linked to antioxidant biochemical pathways specifically, and the others are linked to both antioxidant and anti-inflammatory biochemical pathways. Of particular interest is the upregulation of hsa-miR-30a-5p, which correlates with the decrease of catalase expression protein in red blood cells.

Genetic variation influencing hemoglobin levels and risk for anemia across populations.

Hemoglobin (Hb) concentration is the outcome of the interaction between genetic variation and environmental factors, including nutritional status, sex, age, and altitude. Genetic diversity influencing this protein is complex and varies widely across populations. Variants related to abnormal Hb or altered characteristics of the erythrocytes increase the risk for anemia. The most prevalent are related to the inherited globin abnormalities affecting Hb production and structure. Malaria-endemic regions harbor the highest frequencies of variants associated with the most frequent monogenic diseases and the risk for nonnutritional anemia and are considered as public health problems. Variation in genes encoding for enzymes and membrane proteins in red blood cells also influence erythrocyte life span and risk for anemia. Most of these variants are rare. Interindividual variability of hematological parameters is also influenced by common genetic variation across the whole genome. Some of the identified variants are associated with Hb production, erythropoiesis, and iron metabolism. Specialized databases have been developed to organize and update the large body of available information on genetic variation related to Hb variation, their frequency, geographical distribution, and clinical significance. Our present review analyzed the underlying genetic factors that affect Hb concentrations, their clinical relevance, and geographical distribution across populations.

Advances in understanding the pathogenesis of red cell membrane disorders

Hereditary erythrocyte membrane disorders are caused by mutations in genes encoding various transmembrane or cytoskeletal proteins of red blood cells. The main consequences of these genetic alterations are decreased cell deformability and shortened erythrocyte survival. Red blood cell membrane defects encompass a heterogeneous group of haemolytic anaemias caused by either (i) altered membrane structural organisation (hereditary spherocytosis, hereditary elliptocytosis, hereditary pyropoikilocytosis and Southeast Asian ovalocytosis) or (ii) altered membrane transport function (overhydrated hereditary stomatocytosis, dehydrated hereditary stomatocytosis or xerocytosis, familial pseudohyperkalaemia and cryohydrocytosis). Herein we provide a comprehensive review of the recent literature on the molecular genetics of erythrocyte membrane defects and their reported clinical consequences. We also describe the effect of low-expression genetic variants on the high inter- and intra-familial phenotype variability of erythrocyte structural defects.