Glucose-6-phosphate dehydrogenase (G6PD) is an enzyme presenting in the cytoplasm of human cells that participates in the pentose phosphate pathway and supplies reducing energy by maintaining levels of the co-enzyme nicotinamide adenine dinucleotide phosphate (NADPH). G6PD deficiency is an X-linked inheritance. G6PD gene consists of 13 exons with approximately 18kb and is situated on the distal long arm of X chromosome (Xq28). About 180 mutations of G6PD gene have been reported resulting in protein variants with different levels of enzyme activity. NADPH is essential for both oxidant and antioxidant systems of cells. In the antioxidant pathway, NADPH maintains the reduced form of glutathione to protect cells from oxidative damage. This reduction in cells causes accumulation of redox oxidative species (ROS) and leads to senescence and haemolysis in red blood cell. On the other hand, NADPH is also involved in the oxidant pathway to produce ROS. Although the overproduction of these ROS may adversely affect cell function, cells of immune system also need these reactive species to kill invading organisms. Phagocytes of the immune system need these reactive species to kill invading pathogens as part of the innate immune system. In cases of severe G6PD deficiency, the lack of oxidative metabolism can cause a reduction in oxygen-dependent phagocytosis as observed in chronic granulomatous disease and allows for viral replication [5–8].

Glucose-6-phosphate dehydrogenase (G6PD) deficiency affects more than 400 million people all over the world and prevalence is approximately 35% in Africa and ranges from 6.0 to 10.8% in Southeast Asia. In Myanmar, the prevalence is 11.1% and 4.2% in adult males and females respectively, while it is 15.0% and 2.1% in healthy children males and females respectively. To note, 11.8% of males and 21.0% of females possess the G6PD mutation with the G6PD Mahidol variant occurring in 91.3% of children in Myanmar.

Dengue virus infection is one of the leading causes of morbidity and mortality in children living in tropical and sub-tropical regions. According to WHO, approximately 390 million people worldwide experience a dengue infection annually. In Southeast Asia, dengue leads to over 5,900 deaths annually. In Myanmar, all four dengue serotypes are known to co-circulate, and children are at the highest risk for infection. The reported case fatality rate was 7 per 1,000 dengue cases in 2014.

Most dengue patients present with undifferentiated febrile illness that some may progress to life threatening disease. How patients’ genetic background affects the development of severe infection has become an area of interest. G6PD deficiency is one of the reported genetic variants associated with infections [8, 21]. In vitro studies reported that monocytes from G6PD-deficient individuals had increased susceptibility to dengue virus serotype 2 infections along with higher viral replication [5, 6]. Whether higher replication of dengue virus in G6PD-deficient individuals increases the likelihood of disease severity remains unknown. Herein, we investigated the association between G6PD deficiency and severity of dengue infection in paediatric patients in Myanmar.

The central roles of glucose-6-phosphate dehydrogenase (G6PD) are the production of ribose and the reducing equivalent nicotinamide adenine dinucleotide phosphate (NADPH) via the pentose phosphate pathway (PPP). Both products are vital for the synthesis of many biological building blocks, such as nucleic and fatty acids. It has long been known that NADPH is extremely important in the maintenance of antioxidant defenses. A preponderance of evidence has emerged recently to indicate that NADPH also serves as a pro-oxidant to generate reactive oxygen species (ROS) and reactive nitrogen species (RNS) as signal molecules for promoting cellular processes, such as cell growth. Clinically, G6PD deficiency is the most pervasive X-linked enzymopathy in the world. G6PD-deficient individuals tend to suffer from red cell disorders, including jaundice and drug- or infection-induced hemolytic anemia. These disorders are mostly due to a point mutation in G6PD. Severe G6PD deficiency is intolerant for growth and development in animal models, while a modest increase of G6PD promotes a healthy lifespan.

Many excellent reviews have discussed the pro-survival role of G6PD. How G6PD as a part of PPP affects cells, including cancer cell growth and death, has not been clearly defined. G6PD enhances tumor growth by maintaining intracellular redox homeostasis. G6PD activity is increased in several types of cancers, including bladder, breast, endometrial, esophageal, prostate, gastric, renal, hepatic, colorectal, cervical, lung, and ovarian cancers, glioblastomas and leukemia, as well as gliomas. The current review provides an update of the existing knowledge concerning G6PD and focuses on how G6PD is involved in redox signaling and how it affects cell survival and death, particularly in diseases such as cancer. Exploiting G6PD as a potential drug target against cancer is also discussed.

Severe dengue was not associated with a G6PD deficiency phenotype nor genotype variants whether we used a cut off of < 30% (i.e. only including hemizygous males and homozygous females) or a cut off of < 60%, corresponding to classes I to III of the WHO classification (Table 5).

Severe dengue was diagnosed in 5/29 (17.2%) children with < 60% enzyme activity and in 46/167 (28%) children with G6PD levels > 60% (p = 0.249). In addition, 6/25 (24%) study participants had a G6PD SNP, and 25/103 (24%) did not have a detectable SNP and severe dengue (p = 0.977).

Research and development of a significant number of nucleoside/tide analogs were terminated in the pre-clinical stage due to toxicity in the animal models; though only a few were reported24,46,47 and mitochondrial toxicity was indicated or suspected. To date, due to a number of factors outlined above, mitochondrial toxicity has been difficult to detect in animal species and correlation to humans is at best tenuous.48 Potentially, animal models with genetic mitochondrial abnormalities or humanized animals may be more sensitive to nucleoside/tide toxicity. For example, mice models with genetic mitochondrial abnormalities such as heterozygous superoxide dismutase 2 knockout (Sod+/−) mice were more susceptible to mitochondria toxins than the wild-type animals.49 Recently, Xu et al. reported FIAU-induced liver toxicity in chimeric TK-NOG mice grafted with humanized livers.50 Animal models like these may improve our ability to detect nucleoside toxicity.

To our knowledge this is the first report of MRI findings in a cat with confirmed neuronal ceroid lipofuscinosis. The neuronal ceroid lipofuscinoses represent a heterogeneous group of genetically determined neurodegenerative lysosomal storage diseases. The disease is characterised by abnormal accumulation of autofluorescent lipopigments within the neuronal and extraneural tissue.5 Neuronal ceroid lipofuscinosis has been described in several domestic species, including dogs, cats, cattle, sheep, goats, monkeys and mice.6–13 Clinical signs are similar in all species, including humans, and involve progressive cognitive decline, motor dysfunction, vision deficits and epileptic seizures, ultimately resulting in death or euthanasia. Symptomatic management is generally the mainstay of treatment.14 Encouraging results have, however, been seen using enzyme-replacement therapy and gene therapy.15 Carnitine is a breakdown product of subunit c protein, a major component of the storage material that accumulates in a number of neuronal ceroid lipofuscinoses. Delays in cognitive dysfunction have been reported in English Springer Spaniels supplemented long term with carnitine.15 However, despite these therapeutic advances, neuronal ceroid lipofuscinoses remain an incurable group of diseases.

The cat in this case demonstrated generalised and symmetrical brain cortical atrophy and secondary dilation of the ventricular system and intracranial subarachnoid space (hydrocephalus ex vacuo). These MRI findings have been consistently described in humans and dogs and are considered cardinal signs for neuronal ceroid lipofuscinosis.10,16–19 The cat in this report also showed moderate meningeal thickening. This finding, not described in the human form of the disease, was also reported in three Chihuahuas and a Dachshund with neuronal ceroid lipofuscinosis.9,20 An autoimmune cause of meningeal thickening has been alluded to; however, in the case of the Dachshund, a subdural haematoma was also identified and the findings of thickened meninges could represent reactive meningitis. In our case, the moderate meningeal thickening was due to the diffuse moderate hypertrophy and fibrosis, and could be interpreted as an adaptive change due to brain atrophy. Diffuse T2W white matter hyperintensity with reduction in the white/grey matter distinction has not been previously described in the veterinary literature. A possible explanation for this could be that animals previously reported were scanned with lower field strength MRI magnets. Better contrast resolution of higher field magnets and newer MRI systems could account for this new finding. In humans, periventricular white matter T2W hyperintensity is a common finding in neuronal ceroid lipofuscinosis, confirmed as gliosis and demyelination on histology. In one study, the authors also described a diffuse increase in T2W signal intensity of the white matter.21 Our patient showed a thinned and partially visualised corpus callosum, which, in this case, likely resulted from the generalised brain atrophy. Atrophy of the corpus callosum has also been described in dogs and humans with neuronal ceroid lipofuscinosis.18,22 The cat in our report had generalised calvarial hyperostosis. No skeletal abnormality has been previously described in the human or animal form of the disease. We believe calvarial hyperostosis developed in response to the chronic ex vacuo negative pressure induced by the brain cortical atrophy. Gross and histopathological findings were consistent with previous cases of neuronal ceroid lipofuscinosis in cats,1–4 and confirmed the presence of lysosomal storage neuronal disease with autofluorescent material. TEM showed the presence of characteristic small curvilinear lamellar stacks and electron-dense granular material, consistent morphologically with previously described intra-neuronal lipofuscins in cats, making the diagnosis of neuronal ceroid lipofuscinosis highly likely in this case. Ultrastructural intraneuronal deposits consistent with lipofuscins in cats have been described as granular osmophilic deposits (electron-dense granules), curved lamellar stacks and fingerprint profiles, although this latter, ultrastructural organisation was not observed in our case.

The cat in this case, and those reported in previous studies,1,2,11 presented with neurological features in common with neuronal ceroid lipofuscinosis seen in other species, including humans. Initial manifestations of neuronal ceroid lipofuscinosis in humans occur typically between birth and young adulthood. This is mirrored in dogs and cats, with most reported cases presenting under 2 years of age, as seen in this cat. A case series of feline neuronal ceroid lipofuscinosis in three cats reported disorientation, focal (facial twitching) and generalised epileptic seizures, tactile hyperaesthesia and blindness; signs seen in this cat.4 Similar signs are seen in humans, with a combination of retinopathy, dementia and epilepsy almost always being present.23 In this case no lesions were observed bilaterally in the retina and therefore the blindness reported in this cat was likely central in origin.

To date, 14 different genes have been implemented in the development of the human form of neuronal ceroid lipofuscinosis (CLN1–CLN14). Traditionally and still commonly encountered in the literature, nomenclature was based on the age of clinical presentation, for example infantile/juvenile/adult. The storage material, along with its ultrastructure, is variable for each neuronal ceroid lipofuscinosis type, and, although similar, the clinical course is slightly altered for each one. Molecular analysis has identified the genetic loci for the neuronal ceroid lipofuscinosis types in humans, with the exception of the CLN9 gene, which remains elusive.23 The differences in the mutations account for the variable phenotypes between the types.24 Mutations in nine different genes have been identified in dogs, eight of which are orthologues to the human causative mutations.23,25–28 Sequencing of the exons of CLN1, CLN3, CLN5, CLN8 and CLN10 in a confirmed case of feline neuronal ceroid lipofuscinosis failed to identify the molecular cause in that patient.4 The genes involved in the development of feline neuronal ceroid lipofuscinosis remain unidentified.

In humans and dogs, neuronal ceroid lipofuscinosis has been shown to be a recessively inherited disease with the progeny of two carrier parents having a one in four chance of developing the disease.14 A second cat, from the same litter as the cat in this case, developed similar clinical signs 4 weeks after its littermate and was euthansed owing to severity of clinical signs. Histopathology and TEM confirmed neuronal ceroid lipofuscinosis in this second cat with central nervous system morphological changes similar to the cat described in this case. MRI was not performed in this second case, but pathological findings and the relationship between the two littermates reinforce the hypothesis of an inherited mutation in cats.3

As most nucleoside/tides analogs need to be transformed into 5’-TP to exert on-target and off-target effects, their TP forms are necessary for elucidating these mechanisms (Table 1). The typical initial enzymatic assay for polymerases is a radioactivity-based polymerization assay, in which the inhibition by nucleotides is measured as an IC50 value. The inhibition IC50 assays can be easily adapted to a 96-well filter binding assay, and multiple compounds can be accessed simultaneously. However, based on inhibition studies alone, one cannot establish whether the nucleotide was actually incorporated. Therefore, a single nucleotide incorporation (SNI) assay can be particularly useful to determine if a NTP analog actually served as a substrate for the polymerases. Under a high concentration of NTP analogs (e.g. 500 µM ribose nucleotide TP (rNTP) or 50–100 µM 2′-deoxyribose nucleotide TP (dNTP)), the relative rate of incorporation of an analog can be compared to that of its natural NTP counterpart. For further mechanistic insight, a pre-steady state kinetic analysis of the NTP incorporation offers detailed kinetic parameters such as pre-steady state polymerization rate constant kpol and NTP dissociation constant Kd. In these studies, incorporation rates are studied using multiple time points and nucleotide concentrations, often using quench-flow techniques, making these studies reagent- and time-consuming. Finally, the mechanism of a nucleotide analog-derived enzyme inhibition can be revealed by an elongation assay, in which a stable elongation complex is formed, and the incorporation of the next few natural NTPs is measured. Normally, an NTP analog can be characterized into one of the three groups: chain-terminators, delayed chain-terminators, or stably incorporated analogs. Stably incorporated NTP analogs are difficult to study for toxicity as they could lead to little or no quantitative changes in DNA, RNA, or protein expression.36

The quality of the above-mentioned biochemical assays relies heavily on the availability of high quality TP active forms, which can be time- and resource-consuming to synthesize, and the TP salts are subject to degradation overtime. At times, certain inorganic contaminants could lead to apparent inhibition of polymerases.

Without knowledge of the causative genes involved in feline neuronal ceroid lipofuscinosis, definitive diagnosis is based on necropsy and histopathology results alone. MRI findings, combined with clinical signs, are the mainstay of the ante-mortem presumptive diagnosis. To support the use of this imaging modality in the ante-mortem diagnosis of feline neuronal ceroid lipofuscinosis, recruitment of more feline subjects with confirmed disease into similar descriptive studies is essential in identifying distinct MRI findings within this population. Owing to the similarities in the MRI findings in this cat and those seen in humans and dogs it could be assumed that the imaging features would be the same or similar in other cases of feline neuronal ceroid lipofuscinosis. It would seem prudent, therefore, to consider neuronal ceroid lipofuscinosis within the list of differentials when faced with a cat showing similar neurological signs as our case, with evidence of generalised cerebral and cerebellar atrophy and diffuse T2W hyperintensity of the white matter on MRI.

The G6PD is a key enzyme in the glucose metabolism. G6PD converts glucose-6-phosphate to 6-phosphogluconolactone combined with the reduction of NADP to NADPH. Glutathione reduced by NADPH is essential for neutralizing the oxidant components. G6PD deficiency is the most common human enzyme defect and 400 million people in the world are affected. G6PD deficiency may clinically manifest as a neonatal jaundice and acute hemolytic anemia following ingestion of fava beans (favism) and certain drugs.

So far, Many studies have been performed so far on the relationship between the G6PD deficiency and the susceptibility to development of sepsis in neonates. In our study, the prevalence of G6PD deficiency in male neonates in both groups of patients and controls was significantly more than in the female neonates of same group. This is explained by the x-linked recessive inheritance of G6PD deficiency, because this pattern of inheritance mostly affects males. Also, the prevalence of G6PD deficiency was significantly higher in patients compared to controls. There are reports of an increased prevalence of G6PD deficiency in association with some of the infections.

As previously mentioned, neonatal sepsis occurs more in males compared to females. The exact mechanism of higher susceptibility of sepsis in male neonates is not entirely clear but some reasons, such as the x-linked genes involved in the immune system and hormonal differences were mentioned. We hypothesized that one of the reasons for the higher prevalence of sepsis among male neonates may be due to G6PD deficiency. Since the G6PD deficiency is the most common genetic defect in the world and its mode of inheritance is x-linked recessive, many male newborns were affected. On the other hand, as this study showed, the frequency of G6PD deficiency in neonates with sepsis was higher than in controls. Therefore, we concluded that the G6PD deficiency is one of the reasons for the higher neonates’ susceptibility of developing sepsis, so, G6PD deficiency can be considered a risk factor of male neonatal sepsis.

The exact mechanisms of increased susceptibility of sepsis in G6PD deficient neonates are not clear and require further investigations, but one hypothesis is that reducing the synthesis of ROS resulting from the lack of NADPH due to G6PD deficiency may be one of these mechanisms. Laboratory studies showed a G6PD deficiency decrease production of NADPH in neutrophils. Cooper and colleagues reported that the G6PD deficiency is associated with the decrease in the production of ROS in WBCs of patients, which increases the overall chance of the infection. The other possible mechanisms that might explain the increased prevalence of sepsis among G6PD-deficient neonates was increased serum iron concentration due to lysis of erythrocytes. Some studies showed a relationship between the elevation of blood iron (hyperferremia) and serious infections. For example, the increased prevalence of sepsis in the meningitis patient as a result of intramuscular injection of iron was reported.

In contrast, other studies showed that the incidence of sepsis in premature infants with a gestational age of 27-32 weeks was higher than term infants and it was not related to the G6PD deficiency in patients. Ardati and colleagues concluded in their research that the reduced activity of G6PD to as low as 23% of normal, does not affect the neutrophil function. Also Zareifar and colleagues investigated the prevalence of G6PD deficiency in neonates with sepsis and reported that the G6PD deficiency was not associated with the risk of sepsis infection.

The reduction in the number of leukocytes (leukopenia) and neutrophils (neutropenia) is one of the non-specific symptoms of sepsis. In general, the number of leukocytes increased during sepsis (leukocytosis), but in some cases, the number of leukocytes is reduced. This study showed that the number of the WBCs in the G6PD deficient patients was lower compared to the patients without enzyme deficiency; however, this decrease was not significant. Hsieh and colleagues demonstrated that the G6PD deficient epithelial cells infected with Staphylococcus aureus compared to normal infected epithelial cells have an accumulation of more oxidants and a higher apoptosis rate during infection. This may be due to the decrease in NADPH production as a result of G6PD deficiency, which prevents the effective neutralization of oxidants. It can be assumed that during the sepsis, neutrophils phagocytosis of bacteria and respiratory burst reactions occur. However, because of G6PD deficiency, not enough NADPH is produced to neutralize all the oxidants produced. Therefore, these oxidants cause damage and apoptosis of neutrophils. However, due to the small sample size and the lack of measurement of NADPH, this conclusion is not conclusive and the repeating of this study with a larger population and measurement of NADPH levels is recommended.

This survey was performed in the neonatal intensive care unit (NICU) of Imam Reza University Hospital of Kermanshah in the west of Iran. From 76 newborns with sepsis, 41 were males (53.9%) and 35 were females (46.1%); also, 43 had an early-onset sepsis (56.6%) and 33 had a late-onset sepsis (43.4%). Nine species of bacteria were isolated during the study, the number and frequency of each species being shown in Table 1. The frequency of isolated gram-negative compared to the gram-positive bacteria was higher (Table 2). The most frequent isolated bacteria in both groups of patients with and without G6PD deficiency was Staphylococcus aureus. Blood cultures, in which coagulase-negative Staphylococci were not isolated for more than once or separated from the mixture of bacteria, were considered a contamination and excluded from the results. There was a statistically significant correlation between the prevalence of G6PD deficiency and sepsis in the neonates (p=0.03) (Table 3) in this study.

As Table 3 shows, the prevalence of G6PD deficiency compared in the two groups of female neonate patients and female neonate controls, were not statistically significant (p=0.39), but were statistically significant (p=0.03) in male neonates. The mean WBCs in the G6PD-deficient patients and the G6PD normal patients were 11.6 and 10.7 cells/ mcL respectively (p= 0.77); also, the mean neutrophil percentages in G6PD-deficient patients and G6PD normal patients were 54.86% and 53% respectively (p=0.86).

Altered G6PD status is implicated in many cellular pathophysiological processes and diseases, including hypoxia, inflammation, microbial infection, sepsis, pulmonary vessel dilation, diabetes, hypertension, kidney disease, and brain injuries. The PPP and glutathione-associated metabolic pathways are major antioxidant defense systems in cells. The regulation of these enzymes profoundly affects the development and clinical outcome of diseases.

One of the pro-inflammatory conditions leading to vascular injuries is hyperglycemia. The pro-inflammatory cytokine IL-1β primes high glucose-induced vascular inflammation. In human aortic smooth muscle cells (HASMC), surplus glucose uptake can be activated by IL-1β. Upregulation of the glucose transporter, GLUT-1 or downregulation of mitochondrial respiration alone is insufficient for stimulating the inflammatory response. IL-1β activates the PPP, where excess glucose reroutes to this pathway. This in turn overactivates NOX, which produces superoxide and its reaction with neighboring molecules such as NO, resulting in the production of free radicals that stimulate a downstream inflammatory signaling pathway that leads to endothelial dysfunction. Chronic inflammation in adipose tissue is implicated in insulin resistance found in obesity. Downregulation of G6PD, 6-phosphogluconate dehydrogenase (6PGD) and glutathione-S-transferase (GST) is found in the liver of aged and streptozotocin-induced diabetic rats. The antioxidant, SMe1EC2, increases the G6PD activity but not 6PGD and GST in diabetic rats. SMe1EC2 can also enhance G6PD activity in the lung and heart of aged diabetic rats. These findings suggest that diabetes-induced glucotoxicity can be affected by modulating the activity of redox enzymes.

Diabetes is a condition that impairs the body’s ability to process blood glucose, including type 1, type 2, and gestational diabetes. G6PD deficiency could be a risk factor for diabetes. Impaired G6PD activity by high glucose concentrations in endothelial and kidney cells is associated with increased ROS production and decreased cell survival. A decrease in G6PD expression and activity induced by ubiquitination and an increase in ROS in podocytes occurs at high glucose concentrations. Hyperglycemia in obese mice results in increased oxidative stress in vascular endothelial cells and causes cardiovascular complications. Smaller islets and impaired glucose tolerance are observed in G6PD-deficient mice. Abnormal G6PD status mediates insulin resistance through oxidative stress in adipose tissue found in obese mice. Upregulation of G6PD occurs in pancreatic β-cells in diabetic murine models. Bone marrow transplantation from G6PD-deficient mice to wild-type mice reduces obesity-induced inflammation in adipose tissue and improves insulin resistance. Overexpression of G6PD enhances ROS production and prooxidant enzymes, including iNOS and NOX.

ROS accumulation and β-cell apoptosis are indicative of the development of type 2 diabetes. In high-fat-diet (HFD)-induced obesity, G6PD-deficient mice have decreased insulin and Homeostatic Model Assessment for Insulin Resistance (HOMA-IR) index. These G6PD-deficient mice exhibit glucose and insulin tolerance as well as reduced insulin signaling, suggesting that G6PD deficiency is associated with an improvement in insulin resistance. Downregulation of NOX and upregulation of antioxidant genes, including catalase and glutathione peroxidase (GPx), are observed in G6PD-deficient mice. Although HFD-induced adiposity and fatty liver are not alleviated, the pro-inflammatory macrophages and cytokines are reduced in the G6PD-deficient mice. G6PD-deficient macrophages have decreased phosphorylation of MAPK, nuclear translocation of NF-κB, pro-inflammatory cytokines, and ROS accumulation. These events lead to enhanced insulin sensitivity in G6PD-deficient adipocytes and hepatocytes. Aberrant G6PD status in lipid-overload hepatocytes is associated with impaired pro-inflammatory cytokines via NF-κB and oxidative stress. However, in some diseases with highly inflammasome activation, such as systemic lupus erythematosus, G6PD is upregulated (from array data GDS4185).

Microbial infection in G6PD-deficient patients is mainly related to hemolytic anemia caused by plasmodia or viruses. Lack of G6PD activity is a risk factor for neonatal sepsis in males. Sepsis-induced systemic inflammation and direct pulmonary injury cause acute lung injury (ALI). In airway epithelial cells (AECs), ALI can induce G6PD activity with the concomitant increase of ROS, nitrotyrosine, and NOX2. A G6PD inhibitor 6-AN suppresses airway inflammation in AECs induced by LPS and the ROS derived from NOX2, as well as increasing glutathione reductase activity. G6PD-knockdown A549 lung carcinoma cells are sensitive to staphylococcal infection, leading to apoptosis and ROS accumulation. G6PD-deficient cells are susceptible to corona virus infection through inflammation and NF-κB signaling. G6PD-deficient monocytes are more sensitive to infection by the dengue virus and have redox imbalance compared to matched control monocytes. G6PD is highly expressed in human liver infected by the Hepatitis B virus (HBV) and HBV-associated cancer. G6PD activity can be stimulated by Hepatitis B virus X protein (HBx) mediated by the activation of the transcription factor, Nrf2. Low G6PD activity in children following acute hepatitis infection may cause high morbidity. Mild hepatic encephalopathy (MHE) is a hallmark of chronic liver failure (CLF). The upregulation of G6PD and nNOS in MHE suggests that there is a role for NO in its pathogenesis. Increased level of nNOS and NO is associated with increased activity of NADPH diaphorase in the cerebellum of CLF rats, where overactivation of G6PD is observed.

NADPH oxidation can regulate vascular muscle relaxation. Dimerization of the 1α form of protein kinase G (PKG1α) induced by thiol oxidation contributes to the relaxation of isolated endothelium-removed bovine pulmonary arteries (BPA) to peroxide and responses to hypoxia. G6PD inhibitors 6-AN and epiandrosterone are associated with enhanced PKG1α. An siRNA against G6PD increases PKG1α dimerization in BPA. Hence, reduced G6PD activity is associated with vasodilation, which may be beneficial in ameliorating pulmonary hypertension. In addition, hypoxia activates G6PD and causes proliferation of the pulmonary artery smooth muscle (PASM) cell by increasing Sp1 and hypoxia-inducible factor 1α (HIF-1α), which synthesize less contractile (myocardin and SM22α) and more proliferative (cyclin A and phospho-histone H3) proteins. Consequently, G6PD overactivation contributes to remodeling of pulmonary arterial and development of pulmonary hypertension.

MitoSOX, MitoTracker green and red, and DCFDA staining were done according to manufacturer’s instructions (Invitrogen). Data were acquired with a FACS Calibur flow cytometer (BD Biosciences).

Islets from IKO and control mice were cultured in Aclar embedding film (2-mm thickness, Electron Microscopy Sciences, PA, USA), fixed in 2.5% glutaraldehyde and 4% sucrose in a 0.05 mol/l phosphate buffer, pH 7.4, and examined with a JEOL 1200EX electron microscope (JEOL, Tokyo, Japan) as described previously.

The endoplasmic reticulum (ER) is an important organelle in eukaryotic cells and plays important roles in protein synthesis, modification and processing, folding, assembly, and the transportation of nascent peptide chains (1, 2). The ER has a strong homeostasis system and the stability of the internal environment is the basis for the ER to achieve its functions (3). Some physiological and pathological conditions, including changes in temperature and pH, the accumulation of damaged DNA, contamination with toxic effluents, and infection with viruses and bacteria can cause ER stress (4). ER stress can be divided into three types, including the unfolded protein response (UPR), the ER overload response, and sterol regulatory elements combined with protein-mediated regulatory responses (5). ER stress usually refers to the UPR, which occurs when the misfolded or unfolded proteins in the ER increase and activate the stress signal that transmits to the nucleus through the ER membrane. Upon ER stress, cells mainly elicit two responses: one leading to cellular survival and the other leading to apoptosis (6). Using the survival pathway, cells conquer such disadvantageous effects and maintain homeostasis through the UPR, inhibiting the transcription of mRNA, enhancing the folding capacity of the ER, and ERAD (ER-assisted degradation) to restore homeostasis (7). Under chronic or overwhelming ER stress, the normal functions of the ER fail to recover, resulting in cellular dysfunction and apoptosis (8).

Many ER stress related diseases have been reported in clinical populations (9, 10). When ER stress occurs with high intensity, or it is prolonged, homeostasis is not restored and apoptosis is induced by ER-related molecules. ER-induced apoptosis occurs via three primary pathways, including the IRE1/ASK1/JNK pathway, the caspase-12 kinase pathway, and the C/EBP homologous protein (CHOP)/GADD153 pathway (11, 12). The IRE1/ASK1/JNK pathway is important for apoptosis in the ER and has been found active during many diseases, such as osteoporosis, urothelial carcinoma (13, 14). The caspase-12 kinase pathway is also involved in many diseases, neonatal hypoxic-ischemic encephalopathy, parkinson's disease, etc. (15–17). The CHOP pathway plays an important role in ER stress-induced apoptosis due to pathogenic microbial infections, neurological diseases and neoplastic diseases.

Although glucose-6-phosphate dehydrogenase (G6PD) deficiency is perhaps the most common sex-linked enzymopathy on earth, the biochemical and physiologic roles of this housekeeping enzyme have not been fully explored. Biochemically, G6PD is well known as the rate-limiting enzyme of the pentose phosphate pathway for regenerating nicotinamide adenine dinucleotide phosphate (NADPH) [[3],,,]. NADPH, an essential cofactor in the redox system, maintains a proper level of reducing equivalence such as reduced glutathione (GSH) and acts as a substrate for NADPH oxidase (NOX) and nitric oxide synthase (NOS), which generate reactive oxygen species (ROS) and nitric oxide (NO), respectively, for a subsequent role in signal transduction. Physiologically, evidence has been emerging to indicate that G6PD deficiency affects glucose metabolism, cell growth, embryonic development, lethality and susceptibility to infections by modulating redox homeostasis.

How G6PD deficiency can disrupt immune responses has not been clearly delineated. Since G6PD plays a vital role in cellular redox homeostasis, this enzyme can influence the redox microenvironment in cells leading to modulation of physiological functions. NOXs are a major source of ROS [[16],,] and are involved in the initiation of cell signaling to modulate inflammatory response and the antimicrobial defense in phagocytes. Some transcription factors, such as NF-κB and AP-1, and certain signal transduction pathway proteins, such as MAPKs, are activated by intracellular ROS to induce inflammatory signaling [[20],,]. Patients with G6PD deficiency or G6PD knockdown cells are more susceptible to pathogen infections, indicating that the immune response is affected by G6PD status.

A key physiological function of the innate immune response is the activation of the inflammasome. This mainly leads to the production of pro-inflammatory cytokines, especially interleukin-1β (IL-1β) and IL-18, in response to invading pathogens. The most common inflammasomes include NLRP1, AIM2, NLRP3, and NLRC4, and are classified by their oligomer composition and different stimuli. Among the inflammasomes, NLRP3 is stimulated by environmental- and pathogen/host-derived factors. The processes mediated by inflammasomes are critical during microbial infections, including the regulation of metabolic processes and mucosal immune responses. The activation of the inflammasome requires strict regulation; otherwise, it leads to many diseases [[29],,,]. How G6PD is involved in the activation of the inflammasome has not been clearly defined.

The activation of the NLRP3 inflammasome is ROS dependent and is mediated by the NOX pathway. Decreased ROS production is observed in G6PD-deficient granulocytes upon lipopolysaccharide (LPS) or phorbol 12-myristate 13-acetate (PMA) stimulation and such abnormality has been attributed to impaired NOX signaling. Increased susceptibility to pathogen infections in G6PD-deficient cells is due to an insufficient ROS-triggered inflammatory response. These findings provide support for the notion that G6PD deficiency impairs ROS production via the NOX signaling pathway. The effect of G6PD on NLRP3 inflammasome activation deserves further attention. In the current study, a decrease in IL-1β was observed in the PBMCs of patients with G6PD deficiency and in G6PD-kd THP-1 cells. This led to an investigation of the role of G6PD in inflammasome activation and its association with the bactericidal effect in phagocytes. Mechanistically, G6PD deficiency provides less NADPH as a substrate for NOX, causing less ROS generation to activate the inflammasome.

The authors declare that there are no conflicts of interest.

Although myricetin occurs throughout the Plant Kingdom, it is produced mainly by members of the families Myricaceae, Anacardiaceae, Polygonaceae, Pinaceae and Primulaceae. This phenolic compound is very common in berries, vegetables, and in teas and wines produced from various plants. It occurs in both the free and glycosidically-bound forms, which include myricetin-3-O-(3″-acetyl)-α-l-arabinopyranoside, myricetin-3-O-(4″-acetyl)-α-l-arabinopyranoside, myricetin-3-O-α-l-rhamnopyranoside, myricetin-3-O-β-d-galactopyranoside, myricetin-3-O- (6″-galloyl)-β-d-galactopyranoside, myricetin-3-O-β-d-xylopyranoside, myricetin 3-O-α-l- arabinofuranoside, myricetin-3-O-(2”-O-galloyl)-α-l-rhamnoside, myricetin-3-O-(3”-O-galloyl)- α-l-rhamnoside and myricetin-3-O-α-l-rhamnoside. Myricetin is poorly soluble in water, i.e., 16.6 µg/mL, but dissolves rapidly when deprotonated in basic aqueous media and in some organic solvents such as dimethylformamide, dimethylacetamide, tetrahydrofuran and acetone. Moreover, degradation of this compound, which is most stable at pH 2, was reported to be both pH and temperature dependent.

The history of myricetin (1) extends back to more than a hundred years. It was first isolated in the late eighteenth century from the bark of Myrica nagi Thunb. (Myricaceae), harvested in India, as light yellow-coloured crystals. Isolation was primarily sparked by interest in the dyeing property of the compound. It was well characterised in a further study of Perkin, who established the melting point as 357 °C and prepared various bromo, methyl, ethyl and potassium analogues. This report also described myricitrin (2), a myricetin glycoside (myricetin-3-O-rhamnoside), for the first time. In a subsequent study, Perkin found that myricetin yields a phloroglucinol and gallic acid upon hydrolysis, which served to confirm its chemical structure.

Myricetin (1) is structurally related to several well-known phenolic compounds (Figure 1), namely quercetin (3), morin (4), kaempferol (5) and fisetin (6). The compound is sometimes referred to as hydroxyquercetin, resulting from its structural similarity to quercetin (3). The nutraceuticals and anti-oxidant properties of myricetin are highly valued. Scientific evidence underscores claims that the compound displays a variety of pharmacological activities, including anti-inflammatory, analgesic, antitumour, hepatoprotective and antidiabetic activities.

The theory of oxidative stress (oxygen-free radicals) existed since the last 60 years. However, extensive research in the last three decades has clarified myriads of misconceptions and explored leading roles of oxidative stress in the pathogenesis of many viral diseases [1, 2]. A wide range of the reactive species (RS) is produced as a result of the metabolic process in the body. These RS can be reactive oxygen species (ROS) or reactive nitrogen species (RNS). Previously, RS were only considered to be toxic compounds: however, recent studies have highlighted their involvements in complex cellular signaling pathways and have improved their importance in several biological systems.

The ROS play vital roles in the signaling pathways, cytokine transcription, immunomodulation, ion transport, and apoptosis [4, 5]. Production of the ROS from activated innate immune cells such as neutrophils and macrophages is involved in the destruction of microbes/viruses and infected cells by oxidative bursts. These ROS guide the development of adoptive immune responses, including the proliferation of T cells and positive mediation of B cell functions [7, 8].

Importantly, due to the availability of high-tech facilities, commercial poultry is reared in extensive production systems and therefore is under constant threats to pathogens including viruses. These viruses can infect primarily healthy birds and occasionally vaccinated flocks and cause an irreversible damage to different body tissues. Several viral diseases affect the production of the ROS [10–12], and overproduction of ROS may cause the damage to DNA, protein, and lipid structures, leading to the disruption of the cell functions. This imbalance in the production and detoxification of the ROS is collectively referred as oxidative stress. This review aims at highlighting the molecular mechanisms of oxidative stresses, deleterious effects on cell functions, and their roles in the pathobiology of avian viral infections.

Myricetin is a key ingredient in many foods and is used as a food additive as a result of its anti-oxidant activity and ability to protect lipids against oxidative damage. Available literature portrays the compound as a wonder nutraceuticals and there is no doubt that the molecule holds potential to protect against life threatening diseases, including cancer. The most noteworthy of the biological activities is the protective effect of the compound towards diseases affecting the elderly, such as PD and AD. Although, myricetin alone displays a variety of activities, it seems that its activity may be considerable enhanced through additive or synergistic interactions when in combination with other bioactive compounds. Various researchers have demonstrated its protective nature towards skin aging, suggesting that the compound could be used in cosmetic preparations. In addition, its periphery analgesic effect by enhancing calcium depended potassium channel current and inhibiting excitability of small neurons of dorsal root ganglion in in vivo models supports its role in pain and inflammation. Hence, based on the results from various in vivo studies, myricetin can be developed as an anti-inflammatory and analgesic agent in near future.

However, it has been reported that carbohydrates, DNA and other non-lipid elements in food are degraded by this molecule. Hakkinen and coworkers found that myricetin is susceptible to degradation, since it is labile at high temperatures and is sensitive to certain pH conditions. Food processing and storage is also known to affect the available concentration. These factors should be taken into account before myricetin is used in particular formulations.

The compound has been found to be non-toxic in several in vivo models, although Canada and coworkers reported a degree of toxicity to intestinal cells. This molecule also exerts pro-oxidant effects at higher concentrations. These findings suggest that more toxicity studies should be undertaken before myricetin is included in nutraceuticals and cosmetic preparations.

It has been shown that there is a central role for the Na/K-ATPase oxidant amplification loop in the pathogenesis of obesity as well as the commonly associated comorbidities. The specific mechanism is yet to be elucidated, but the role of HO-1 upregulation has also been characterized. Both of these signaling mechanisms present interesting therapeutic strategies for the treatment of obesity, NASH and cardiovascular diseases, while offering the possibility of limiting off-target effects. By increasing our understanding of the molecular biology and cellular mechanics involved we will not only gain an understanding of the exact sequence of events that precede these diseases, but also potentially have new biomarkers that act as a “warning system” for these diseases. Utilization of genetic therapies that specifically target the tissue of interest, as in the lentiviral gene therapy models, allows the opportunity for specific expression in target tissues, which limits off-target effects. Understanding the mechanics of a signaling pathway and its downstream targets would improve the understanding of disease progression.

HepG2 cells were treated in sodium pyruvate-containing Dulbecco modified Eagle medium supplemented with 10% dialyzed FBS (Life Technologies), 50 μM uridine (Alfa Aesar, Haverhill, MA), l-glutamine, and penicillin-streptomycin. Cells were seeded at 4 × 104 cells/well in 2 ml of media per well in 12-well plates 1 day before the addition of the test compounds. CEM cells were treated in RPMI 1640 media supplemented with 10% dialyzed FBS, 1 mM sodium pyruvate (Life Technologies), 50 μM uridine (Alfa Aesar), l-glutamine, and penicillin-streptomycin. CEM cells were seeded at 2 × 104 cells/ml in 2 ml of media per well in 12-well plates on the day of the addition of drug. Cells were cultured continuously in 5% CO2 for 14 days in the presence of compounds. For HepG2 cells, the medium was changed on day 6 and day 9 to media with fresh drug. CEM cells were passaged by 1:10 dilution on days 6 and 9 in media with fresh drug. On day 14, the cell-free culture medium was collected. Cells were collected and counted using disposable hemocytometers (INCYTO, Covington, GA). Each experiment was repeated three times with each repeat initiated on different days. The lactate concentration in cell-free medium was measured by using an EnzyChrom l-lactate assay kit (BioAssay System, Hayward, CA). Briefly, all of the samples were diluted 20-fold with water, and the lactate concentration was measured according to the manufacturer’s instructions. The lactate concentration was expressed as a percentage of the mock-treated controls. The effect of drugs on mitochondrial DNA quantity per cell was measured by comparing the relative mitochondrial genome copy number (mtDNA copy number divided by nuclear DNA copy number) in cells treated with drug versus mock-treated cells. A nuclear DNA target sequence was used that corresponds to the β-actin gene (38), and a mitochondrial target sequence was used that corresponds to mitochondrial DNA nucleotide positions 10620 to 10710 (39). The primers and TaqMan probes (Eurofins MWG Operon, Louisville, KY) used for real-time PCR were ordered were as follows. The primers and probe used for the quantification of nuclear DNA (β-actin gene) were the sense primer 5′-GCGCGGCTACAGCTTCA-3′, the antisense primer 5′-TCTCCTTAATGTCACGCACGAT-3′, and the probe 5′-(FAM)-CACCACGGCCGAGCGGGA-(BHQ)-3′. The primers and probe for the quantification of mtDNA were the forward primer MH533 (5′-ACCCACTCCCTCTTAGCCAATATT-3′), the reverse primer MH534 (5′-GTAGGGCTAGGCCCACCG-3′), and Mito-Probe [5′-(FAM)-CTAGTCTTTGCCGCCTGCGAAGCA-(BHQ)-3′]. The cells were collected after 14 days of drug treatment and pelleted by centrifugation at 8,600 × g for 2 min. The cell pellets were resuspended in PBS, and the total DNA was isolated by using a DNeasy tissue kit (Qiagen, Hilden, Germany). Cellular DNA from mock-treated cells was serially diluted and used to generate corresponding standard curves for determining a relative copy number of the gene targets. qPCR of each sample was performed in triplicate in a 7500 real-time PCR system (Applied Biosystems, Foster City, CA) in a 15-μl total reaction volume containing 1× PCR mix (Life Technologies, Carlsbad, CA), 200 nM β-actin probe (for nuclear DNA) or Mito-Probe (for mtDNA), 750 nM β-actin sense and antisense primers (for nuclear DNA) or MH533/MH534 primers (for mtDNA), and 1.5 μl of a purified, serially diluted DNA sample. The mitochondrial and cellular genome copy numbers were calculated based on the standard curves, and the relative mitochondrial copy number/cell was calculated by dividing the measured mitochondrial copy number by the copy number of the β-actin gene. Changes in the mitochondrial copy number/cell were expressed as a percentage of the mock-treated control.

Staged young adults of mock and G6PD-knockdown C. elegans were harvested and treated with various concentrations of H2O2 prepared in PBS for 30 min at room temperature on a test tube rotator (Snijders, Tilburg, Netherlands). Immediately after the treatment, the worms were pelleted and rinsed with PBS to remove the residual H2O2. The treated C. elegans was transferred to fresh RNAi NGM plate for recovery for 2 h followed by picking hermaphrodites for scoring apoptotic germ cells and egg production.

As the prevalence of obesity increases, it is paramount to note that the incidence of comorbidities, such as metabolic syndrome, NASH and cardiovascular disease, are going to increase as well. Obesity is associated with systemic oxidative stress, and it is suggested that impaired mitochondrial function and severe inflammation in the adipocyte underlie the pathogenesis of obesity. Hypertriglyceridemia and hyperglycemia, both associated with increased adiposity, have been shown to increase plasma FFA, which can lead to increased ROS generation and oxidative stress; this imbalance and oxidative stress has been implicated in both NASH and cardiovascular diseases. Understanding the role of ROS and oxidative stress in these disease states is a crucial step towards better therapeutic strategies. To this end, recent advancements in the understanding of both the Na/K-ATPase oxidant amplification loop and HO-1, provide a unique opportunity to better elucidate the redox mechanisms that modify inflammation and manipulate localized redox signaling pathways. It is highly possible that the mechanisms operant in both the Na/K-ATPase oxidant amplification loop and HO-1 may also involve other mediators (as yet unidentified) that directly modulate cellular oxidative and inflammatory responses. Although, the pharmacological and non-pharmacological interventions targeting these mechanisms have been demonstrated to be effective, it is possible that implementation of other strategies or targeting other pathways might prove to have better clinical outcomes. However, the success of these strategies will open up new avenues and approaches toward the antagonism of obesity and subsequent NASH, once we bridge the gap from mice to humans.

Infectious bronchitis virus (IBV) causes infectious bronchitis in poultry and is endemic in all poultry-producing regions of the world. The IBV virulence affects the oxidative status by differentially modulating MnSOD. Highly virulent strain significantly increases the level of MnSOD than an attenuated virus. Increased level of MnSOD may direct the more significant immune response to eradicate the virus. The same group of researchers also demonstrated that IBV infection increases the abundance of glutathione S-transferase 2, a protein of the sulfotransferase family, and L-lactate dehydrogenase.

In recent years, berberine has been demonstrated to treat DM by modulating the structure and diversity of gut microbiota, including enrichment of beneficial microbes and inhibition of harmful microbes (Liu L et al. 2010). The bioavailability of berberine is very low, and the absorption rate is only 5–10% in the intestinal tract. However, it can significantly reduce the activity of disaccharidase and α-glucosidase in the intestinal tract, resulting in a reduction the absorption of glucose and postprandial hyperglycemia (Liu L et al. 2010; Li ZQ et al. 2012). CR alkaloid treatment avoided a decline in the diversity of gut microbes in obese mice and favoured the maintenance of a stable and healthy bacterial community in high-fat high cholesterol (HFHC)-fed animals (Kai 2017). Berberine can lead to an increase in the abundance of probiotics such as Blautia, Bacteroides, Bifidobacteria and Lactobacillus, and a decrease in relative abundance of Firmicutes and Bacteroides in the intestinal tract of animals (Meng et al. 2016; Gu et al. 2017).

Another study showed that the berberine selectively enriched the propionic acid producing bacteria and intestinal barrier repair bacteria Ackermansia; a CR decoction promoted butyric acid producing bacteria, such as Coprococcus, Faecalibacterium and Oscillospira. Compared with berberine, the CR decoction induced higher flora diversity, and the flora structure was closer to that of normal animals (Ti 2017). The increase of GLP-1 and short-chain fatty acids in the gut may account for the structural and diversity changes to the microbiota induced by berberine (Sun et al. 2016).

Herbal medicines, including TCMs, are considered useful agents to treat various human diseases (Li et al. 2009; Peng et al. 2018). CR has a long history of being used as an important herbal medicine in Asian countries because of its reliable curative effects against various diseases. Nowadays, the most predominant traditional uses of CR have been confirmed by modern pharmacological research. So far, these investigations have reported that CR contains abundant isoquinoline alkaloids (especially berberine), which are also the active substances responsible for the pharmacological effects of this TCM. CR and berberine have a broad-spectrum antibacterial effect, manifesting as bacteriostasis at low concentrations and sterilization at high concentrations. This suggests that a combination of berberine or CR and conventional antibacterial drugs might exert a greater effect. Intensive research has indicated that CR has potential as a cardioprotective agent. In addition to reducing the incidence, it also protects the heart from MI/R injury. These properties are mainly attributed to berberine, coptisine, palmatine, epiberberine, jatrorrhizine and magnoflorine. Many studies have demonstrated modulation of the composition of the gut microbiota (enrichment of beneficial microbiota and inhibition of harmful microbiota) as one of the most important aspect for treating obesity, diabetes, and other metabolic disorders. As a natural compound with both anti-inflammatory and antitumor activities, berberine shows great potential in cancer treatment. However, the effects of berberine are not strong; therefore, structural modification of berberine is required. Moreover, CR containing various active components may be more effective than its single component berberine and could provide multiple therapeutic effects. There is a significant difference between the blood concentration and the tissue concentration. Therefore, to find a suitable pharmacokinetic marker for CR may be challenging but is necessary. Moreover, the pharmacokinetics of TCM should try to elucidate all the chemical components entering the body and their processes in the body (absorption, distribution, metabolism and excretion), with the aim of building a bridge between the complex chemical components and the systemic clinical effects, to reveal the underlying mechanism(s). Additionally, related target-organ toxicity evaluations are lacking. Thus, more work should be devoted to investigating the pharmacokinetics and features of CR and its active components, and further clinical studies are required to evaluate the potential curative effects and possible toxicities of CR and its active components toward the target organs. In addition, according to the current pharmacological research, berberine is not only the main active component but also the primary toxic component of CR. Consequently, it is crucial to develop a strategy to balance the pharmacological effects and toxicity of berberine. Besides, current reports on the original plants used to make CR, including C. chinensis, C. deltoidea and C. teeta, commonly focus on the chemical components and pharmacological effects of the roots because of their traditional use in TCM, and the other parts of the plants are often ignored and disposed of without pretreatment (Shen 2006). However, some previous reports revealed that the leaves of the CR plants also contain berberine (Li et al. 2004; Liu T et al. 2010). Therefore, further research is required to investigate the chemical constituents and pharmacological activities of the other parts of the original CR plants.

This present study systematically reviewed the traditional uses, botany, phytochemistry, pharmacology, and toxicology of CR to provide comprehensive information regarding this herbal medicine, which could be beneficial for highlighting the importance of CR and providing some clues for the future research of this herbal medicine.